Effect of cell appendages on the adhesion properties of a highly adhesive bacterium, Acinetobacter sp. Tol 5

A toluene-degrading bacterium, Acinetobacter sp. Tol 5, shows noteworthy adhesiveness mediated by two types of cell appendages. In this study, we obtained a less-adhesive mutant, T1, which lost both types of appendages, and investigated how the cell appendages affect the adhesion properties of this useful bacterium for environmental technology. Wild-type cells attained irreversible adhesion to polyurethane carriers within 30 s, while adhesion of T1 cells was still reversible at that time. While T1 showed decreased adhesion with decreasing ionic strength and did not adhere at all at 0.015 mM, adhesion of the wild type was fully independent of ionic strength. Acinetobacter sp. Tol 5 was also found to be not motile. Our results suggest that through the long distant interaction mediated by the appendages between the cells and surfaces, Tol 5 cells can attain irreversible adhesion very quickly without approaching the vicinity of the substratum.     

Hydrophobic interactions of group A streptococci with hexadecane droplets.

The adherence of Streptococcus pyogenes cells to hexadecane droplets was measured by vortexing water suspensions of streptococci with hexadecane. It was found that adherence of the organisms to hexadecane droplets was abolished by pretreating the organisms with trypsin, pepsin at pH 4.5, or HCl solutions at 95 degrees C. Streptococcal adherence was best expressed in organisms harvested during the stationary phase of growth and was inhibited by fatty acid-free albumin because of the interaction of the protein with the streptococcal surfaces. The data suggest that adherence to hexadecane droplets measures the availability on the surface of S. pyogenes cells of lipophilic residues that are either hydrophobic regions of surface protein structures or, more likely, glycolipids complexed with and oriented by surface proteins.     

Effect of Cell Appendages on the Adhesion Properties of a Highly Adhesive Bacterium,Acinetobacter sp. Tol 5

A toluene-degrading bacterium, Acinetobacter sp. Tol 5, shows noteworthy adhesiveness mediated by two types of cell appendages. In this study, we obtained a less-adhesive mutant, T1, which lost both types of appendages, and investigated how the cell appendages affect the adhesion properties of this useful bacterium for environmental technology. Wild-type cells attained irreversible adhesion to polyurethane carriers within 30 s, while adhesion of T1 cells was still reversible at that time. While T1 showed decreased adhesion with decreasing ionic strength and did not adhere at all at 0.015 mM, adhesion of the wild type was fully independent of ionic strength. Acinetobacter sp. Tol 5 was also found to be not motile. Our results suggest that through the long distant interaction mediated by the appendages between the cells and surfaces, Tol 5 cells can attain irreversible adhesion very quickly without approaching the vicinity of the substratum.     

Cell-surface hydrophobicity of adherent oral bacteria

Adherent bacteria were released from the surfaces of four freshly extracted teeth by mild sonic oscillation, and screened for cell-surface hydrophobicity on the basis of their ability to adhere to hexadecane. Of the 103 tooth isolates examined, 82 adhered to the test hydrocarbon. Hydrophobic bacteria could similarly be isolated from the stainless steel dental matrix bands following brief incubation in the mouth of a volunteer; 30 of 52 isolates examined adhered to hexadecane. Among those strains which adhered to hexadecane, streptococci were the most frequent type isolated. Various other morphological types were also observed, including cocci, bacilli, coryneforms, and filamentous bacteria. The high overall proportion of hydrophobic bacteria found in this study (72%) suggests that cell-surface hydrophobicity may play a role in adherence of certain oral species to the tooth surface.     

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.     

Comparison of contact angles and adhesion to hexadecane of urogenital, dairy, and poultry lactobacilli: effect of serial culture passages.

The aim of this study was to examine the hydrophobicities of 23 urogenital, dairy, poultry, and American Type Culture Collection isolates of lactobacilli and to determine the effect on hydrophobicity of serially passaging the strains in liquid medium. To this end, strains were grown after isolation and identification and then serially passaged up to 20 times. Hydrophobicity was assessed through contact angle measurements on lawns of cells by using water, formamide, methylene iodide, 1-bromonaphthalene, and hexadecane as wetting agents and through measurement of their partitioning in a hexadecane-water system. The hydrophobicities of these strains varied widely, with Lactobacillus casei strains being predominantly hydrophilic and L. acidophilus strains being mostly hydrophobic. For some isolates, serial passaging was accompanied by a clear loss of hydrophobic surface properties, whereas for other strains, cultures became heterogeneous in that some cells had already lost their hydrophobic surface properties while others were still hydrophobic. Adhesion of this collection of lactobacilli to hexadecane droplets in microbial adhesion to hexadecane (MATH) tests was driven by their aversion to water rather than by their affinity for hexadecane, as concluded from the fact that hexadecane contact angles were zero for all strains. Furthermore, adhesion of the lactobacilli to hexadecane in MATH tests occurred only when the water contact angle on the cells was above 60 degrees.     

Comparison of contact angles and adhesion to hexadecane of urogenital, dairy, and poultry lactobacilli: effect of serial culture passages.

The aim of this study was to examine the hydrophobicities of 23 urogenital, dairy, poultry, and American Type Culture Collection isolates of lactobacilli and to determine the effect on hydrophobicity of serially passaging the strains in liquid medium. To this end, strains were grown after isolation and identification and then serially passaged up to 20 times. Hydrophobicity was assessed through contact angle measurements on lawns of cells by using water, formamide, methylene iodide, 1-bromonaphthalene, and hexadecane as wetting agents and through measurement of their partitioning in a hexadecane-water system. The hydrophobicities of these strains varied widely, with Lactobacillus casei strains being predominantly hydrophilic and L. acidophilus strains being mostly hydrophobic. For some isolates, serial passaging was accompanied by a clear loss of hydrophobic surface properties, whereas for other strains, cultures became heterogeneous in that some cells had already lost their hydrophobic surface properties while others were still hydrophobic. Adhesion of this collection of lactobacilli to hexadecane droplets in microbial adhesion to hexadecane (MATH) tests was driven by their aversion to water rather than by their affinity for hexadecane, as concluded from the fact that hexadecane contact angles were zero for all strains. Furthermore, adhesion of the lactobacilli to hexadecane in MATH tests occurred only when the water contact angle on the cells was above 60 degrees.     

Physicochemical and structural studies onAcinetobacter calcoaceticus RAG-1 and MR-481—Two standard strains in hydrophobicity tests

Acinetobacter calcoaceticus RAG-1 and MR-481, two standard strains used in microbial adhesion to hydrocarbons (MATH), were characterized by contact angles, pH-dependent zeta potentials, elemental surface composition by X-ray photoelectron spectroscopy (XPS), and molecular composition by infrared spectroscopy (IR). Negatively stained (methylamine tungstate) and ruthenium red-stained cells were studied by transmission electron microscopy to reveal the absence or presence of surface appendages. Despite the fact thatA. calcoaceticus RAG-1 is known to be extremely hydrophobic in MATH, whereas MR-481 is a completely non-hydrophobic mutant, neither XPS nor IR indicated a significant difference in chemical composition of the cell surfaces. Contact angles with polar liquids, water and formamide, were considerably higher on RAG-1 than on MR-481, in accordance with their relative hydrophobicities as measured by MATH. However, no significant differences in contact angles were observed between the two strains with apolar liquids like diiodomethane,-bromonaphthalene, and hexadecane. Fibrous extensions on RAG-1, observed after ruthenium red staining, were absent on the non-hydrophobic mutant MR-481. Tentatively, these extensions could be held responsible for the hydrophobicity ofA. calcoaceticus RAG-1.     

Role of Thin Fimbriae in Adherence and Growth of Acinetobacter calcoaceticus RAG-1 on Hexadecane

Acinetobacter calcoaceticus RAG-1, a hydrocarbon-degrading bacterium which adheres avidly to hydrocarbons and other hydrophobic surfaces, possesses numerous thin fimbriae (ca. 3.5-nm diameter) on the cell surface. MR-481, a nonadherent mutant of RAG-1 which is unable to grow on hexadecane under conditions of limited emulsification and low initial cell density, lacks these fimbriae. Prolonged incubation of MR-481 in hexadecane medium enriched for partial adherence revertants. The reappearance of thin fimbriae was observed in all such revertant strains. RAG-1 cells and partial revertant strains were agglutinated in the presence of antibody, whereas MR-481 cells were not. Another mutant, AB15, which was previously isolated on the basis of its nonagglutinability in the presence of antibody, also lacked thin fimbriae and was conditionally nonadherent. Furthermore, strain AB15 was unable to grow on hexadecane medium. Adherence of RAG-1 cells to hexadecane was considerably reduced after shearing treatment. The material removed from the cell surface by shearing of RAG-1 and the partial revertant strains yielded a single antigenic band in RAG-1 and partial revertant strains, as observed by crossed immunoelectrophoresis. This band was absent in both fimbriae-less mutants, MR-481 and AB15. The data demonstrate that the thin fimbriae of RAG-1 (i) are a major factor in adherence to polystyrene and hydrocarbon, (ii) may be crucial in enabling growth of cells on hexadecane, and (iii) constitute the major cell surface agglutinogen.     

Use of monoclonal antibodies to demonstrate different sites with different functional characteristics in a bacterial lipase from Pseudomonas aeruginosa YS-7.

Structural and functional features of the extracellular lipase from the low-water-tolerant bacterium Pseudomonas aeruginosa YS-7 were studied immunochemically with the aid of monoclonal antibodies (MAbs) raised against the enzyme. Fourteen different MAbs were obtained, verified as immunoglobulin G types, and characterized by their interaction with the enzyme in relation to (i) inhibition of activity of free enzyme, (ii) inhibition of activity of adsorbed enzyme, (iii) interaction with the cell-bound enzyme, and (iv) inhibition of adherence to hexadecane droplets. Four of the MAbs exhibiting the highest binding constants (Kapp greater than 10(8) M-1) were selected for further study of the lipase. Their binding to the enzyme was assayed by means of adapted enzyme-linked immunosorbent assay techniques. Use of these MAbs in single or dual binding procedures made it possible to reveal several distinct sites on the lipase macromolecule. Two of these are functional sites, one for hydrophobic adhesion (binds MAb 5) and the other (binds MAb 1) for implementation of its hydrolytic activity. A third binding site (binds MAb 8) does not participate directly in either of the above functions. A fourth binding site (binds MAb 10) appears to be involved in the active expression of the enzyme. The cell-associated form of the lipase seems to be located on the external surface of the cells with its active site exposed. It appears to be anchored to the outer membrane of the cells by means of its hydrophobic region in a way that resembles its adherence to hydrophobic surfaces such as hexadecane droplets.     

Use of monoclonal antibodies to demonstrate different sites with different functional characteristics in a bacterial lipase from Pseudomonas aeruginosa YS-7.

Structural and functional features of the extracellular lipase from the low-water-tolerant bacterium Pseudomonas aeruginosa YS-7 were studied immunochemically with the aid of monoclonal antibodies (MAbs) raised against the enzyme. Fourteen different MAbs were obtained, verified as immunoglobulin G types, and characterized by their interaction with the enzyme in relation to (i) inhibition of activity of free enzyme, (ii) inhibition of activity of adsorbed enzyme, (iii) interaction with the cell-bound enzyme, and (iv) inhibition of adherence to hexadecane droplets. Four of the MAbs exhibiting the highest binding constants (Kapp greater than 10(8) M-1) were selected for further study of the lipase. Their binding to the enzyme was assayed by means of adapted enzyme-linked immunosorbent assay techniques. Use of these MAbs in single or dual binding procedures made it possible to reveal several distinct sites on the lipase macromolecule. Two of these are functional sites, one for hydrophobic adhesion (binds MAb 5) and the other (binds MAb 1) for implementation of its hydrolytic activity. A third binding site (binds MAb 8) does not participate directly in either of the above functions. A fourth binding site (binds MAb 10) appears to be involved in the active expression of the enzyme. The cell-associated form of the lipase seems to be located on the external surface of the cells with its active site exposed. It appears to be anchored to the outer membrane of the cells by means of its hydrophobic region in a way that resembles its adherence to hydrophobic surfaces such as hexadecane droplets.     

Unmasking of surface components by removal of cell-associated emulsan from Acinetobacter Sp. RAG-1

Summary Acinetobacter calcoaceticusRAG-1 cells lacking the emulsan capsule on the cell surface were obtained by two methods; a) by selecting for mutants that lack emulsan with a specific phage and b) by removal of the emulsan capsule from wild type cells with a specific emulsan depolymerase. Emulsan deficient cells obtained by either method become deficient in the adsorption of phage ap3 and sensitive to a newly isolated bacteriophage, nø. When RAG-1 cells were first treated with emulsan depolymerase and subsequently incubated without the enzyme, regeneration of the cell-associated emulsan was correlated with an increase in phage ap3 adsorption and an inhibition in phage nø adsorption. By partial regeneration of cell surface emulsan, a physiological state was obtained in which RAG-1 cells were sensitive to and efficiently adsorbed found phages. Enzyme-treated RAG-1 cells were found to be more adherent to hexadecane than the untreated RAG-1 cells. The data indicate that in addition to its function as the ap3 receptor, cell-associated emulsan masks the expression of other cell-surface determinant(s) which function(s) as: (i) receptor for bacteriophage nø, and (ii) cell-surface sites which enhance adherence to hydrophobic surfaces.Present address: Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA     

Involvement of Acinetobacter sp. in the floc-formation in activated sludge process

The coaggregation behavior of Acinetobacter johnsonii S35 isolate with sewage bacteria was assessed by a spectrophotometric assay using different samples from a municipal wastewater treatment plant and a community plant. A. johnsonii S35 coaggregated well with other free bacteria and microflocs at the mixing ratios of 0.2:1-0.6:1 of A. johnsonii S35 and sewage samples. In addition, the size of coaggregates became larger (100 μm or more) under the same conditions. A. johnsonii S35 cells were highly adsorbed (adsorption=93-99%) onto sludge samples. Microbial adhesion to hydrocarbon (MATH) test and adsorption to octyl-Sepharose CL-4B showed that A. johnsonii S35 cells and sludge samples had a hydrophobic character. The population of Acinetobacter spp. in sewage treatment plants was 2-7% and its role in bioflocculation was discussed. The present study revealed that A. johnsonii S35 isolate can play as a bridging organism and contribute in floc-formation in activated sludge process.     

Structural organization of DMPC lipid layers on chemically micropatterned self-assembled monolayers as biomimetic systems

The growth structure of DMPC lipid layers on hydrophobic and hydrophilic alkylsilane-based self-assembled monolayers adsorbed on silicon has been investigated by means of X-ray reflectometry and atomic force microscopy. Hydrophilic modification of hydrophobically terminated ODS-SAMs has been achieved by dose-controlled irradiation with DUV light. While island formation of small DMPC bilayer islands is observed on hydrophobic SAM surfaces, closed layers of DMPC monolayers are formed on hydrophilic SAM surfaces. Furthermore, DMPC adsorption on chemically micropatterned substrates with alternating hydrophobic/hydrophilic surface properties has been studied by imaging ellipsometry and photoemission microscopy. Indication for at least partial bridging of hydrophobic areas by an adsorbed DMPC monolayer has been found.     

Increased cell surface hydrophobicity of a Serratia marcescens NS 38 mutant lacking wetting activity.

The cell surface hydrophobicity of Serratia marcescens appears to be an important factor in its adhesion to and colonization of various interfaces. The cell surface components responsible for mediating the hydrophobicity of S. marcescens have not been completely elucidated, but may include prodigiosin and other factors. In the present report we have investigated the potential role of serratamolide, an amphipathic aminolipid present on the surfaces of certain S. marcescens strains, in modulating cell surface hydrophobicity. The hydrophobic properties of a serratamolide-producing strain (NS 38) were compared with those of a serratamolide-deficient mutant (NS 38-9) by monitoring the kinetics of adhesion to hexadecane. Serratamolide production was monitored by thin-layer chromatography and the wetting activity of washed-cell suspensions on polystyrene. Wild-type NS 38 cells were far less hydrophobic than the serratamolide-deficient mutant cells were; the removal coefficients were 48 min-1 for the mutant, as compared with only 18 min-1 for the wild type. The data suggest that the presence of serratamolide on S. marcescens cells results in a reduction in hydrophobicity, presumably by blocking hydrophobic sites on the cell surface.     

Phenol and <Emphasis Type="Italic">n</Emphasis>-alkanes (C<Subscript>12</Subscript> and C<Subscript>16</Subscript>) utilization: influence on yeast cell surface hydrophobicity

This study was focused on the role of two types of diametrically different carbon sources, n-alkanes represented by a mixture of dodecane–hexadecane, and phenol on modification of the cell surface hydrophobicity. Capabilities of using either solely hydrocarbons or hydrocarbons in the mixture with phenol as well as phenol itself by yeast species Candida maltosa, Yarrowia lipolytica and Pichia guilliermondii were investigated. Studies were complemented by cell biomass formation measurements. The corresponding cell surface hydrophobicity was assessed by microbial adhesion to the hydrocarbon test (MATH). Degradation of phenol was examined using GC-SPE technique, whereas hydrocarbons were extracted prior to gravimetric determination. Results obtained indicated that the hydrophobic or hydrophilic nature of the carbon source had significant influence on the cell surface hydrophobicity. Although the results differed for some individual yeast strains, the generalization can be made that there is the correlation between the best hydrocarbon and phenol degradation and corresponding cell wall properties of the yeast examined.     

Hydrophobic interactions within biofilms of nitrifying and denitrifying bacteria in biofilters

Hydrophobicity of the solid surface and microbial cell surface is important factor for the development of biofilms applied in bioengineering systems. An adsorption of phenanthrene was used for analysis of the hydrophobicity of support fibers and bacterial cell surfaces within the biofilter of wastewater. The adsorption of phenanthrene was measured by synchronous fluorescence spectrometry. Cell surface hydrophobicity does not depend on the fixation procedure, pH of microbial suspension, and has no clear correlation with an adherence of the cells to hexadecane droplets. Notwithstanding high hydrophobicity of bacterial cells, the hydrophobicity of intact biofilm is determined by the hydrophobicity of the support fibers. New indexes were proposed to evaluate the reactor performance related with hydrophobic interactions within the biofilm. These indexes showed that significant share of hydrophobic sites within the nitrifying biofilm is protected from the hydrophobic interactions between the cells and environment.     

Energy of adhesion of human T cells to adsorption layers of monoclonal antibodies measured by a film trapping technique.

A novel method for studying the interaction of biological cells with interfaces (e.g., adsorption monolayers of antibodies) is developed. The method is called the film trapping technique because the cell is trapped within an aqueous film of equilibrium thickness smaller than the cell diameter. A liquid film of uneven thickness is formed around the trapped cell. When observed in reflected monochromatic light, this film exhibits an interference pattern of concentric bright and dark fringes. From the radii of the fringes one can restore the shape of interfaces and the cell. Furthermore, one can calculate the adhesive energy between the cell membrane and the aqueous film surface (which is covered by a layer of adsorbed proteins and/or specific ligands), as well as the disjoining pressure, representing the force of interaction per unit area of the latter film. The method is applied to two human T cell lines: Jurkat and its T cell receptor negative (TCR-) derivative. The interaction of these cells with monolayers of three different monoclonal antibodies adsorbed at a water-air interface is studied. The results show that the adhesive energy is considerable (above 0.5 mJ/m2) when the adsorption monolayer contains antibodies acting as specific ligands for the receptors expressed on the cell surface. In contrast, the adhesive energy is close to zero in the absence of such a specific ligand-receptor interaction. In principle, the method can be applied to the study of the interaction of a variety of biological cells (B cells, natural killer cells, red blood cells, etc.) with adsorption monolayers of various biologically active molecules. In particular, film trapping provides a tool for the gentle micromanipulation of cells and for monitoring of processes (say the activation of a T lymphocyte) occurring at the single-cell level.     

IR study of self-assembly of capsular exopolymers from Pseudomonas sp. NCIMB 2021 on hydrophilic and hydrophobic surfaces

Capsular exopolymers (EPS) of the bacterium Pseudomonas sp. NCIMB 2021 are allowed to self-assemble on hydrophilic and hydrophobic gold surfaces. Tapping mode atomic force microscopy confirms the differences in the surface topography between EPS adsorbed on both surfaces. Fourier-transform IR spectroscopy indicates that the EPS surface coverage is much greater on the hydrophobic surface. Furthermore, an increased contribution is observed from hydrophobic (i.e., methyl and tyrosyl residues) and electrostatic (i.e., carboxylate residues) groups at the hydrophobic surface, but there is relatively less neutral polymer compared to the hydrophilic surface. The behavior of this EPS is in agreement with the behavior of cells of Pseudomonas sp. NCIMB 2021 at hydrophilic and hydrophobic surfaces.     

Conformational changes and orientation of Humicola lanuginosa lipase on a solid hydrophobic surface: an in situ interface Fourier transform infrared-attenuated total reflection study

This study was done to better understand how lipases are activated at an interface. We investigated the conformational and solvation changes occurring during the adsorption of Humicola lanuginosa lipase (HLL) onto a hydrophobic surface using Fourier transform infrared-attenuated total reflection spectroscopy. The hydrophobic surfaces were obtained by coating silicon attenuated total reflection crystal with octadecyltrichlorosilane. Analysis of vibrational spectra was used to compare the conformation of HLL adsorbed at the aqueous-solid interface with its conformation in solution. X-ray crystallography has shown that HLL exists in two conformations, the closed and open forms. The conformational changes in HLL caused by adsorption onto the surface were compared with those occurring in three reference proteins, bovine serum albumin, lysozyme, and alpha-chymotrypsin. Adsorbed protein layers were prepared using proteins solutions of 0.005 to 0.5 mg/mL. The adsorptions of bovine serum albumin, lysozyme, and alpha-chymotrypsin to the hydrophobic support were accompanied by large unfoldings of ordered structures. In contrast, HLL underwent no secondary structure changes at first stage of adsorption, but there was a slight folding of beta-structures as the lipase monolayer became complete. Solvation studies using deuterated buffer showed an unusual hydrogen/deuterium exchange of the peptide CONH groups of the adsorbed HLL molecules. This exchange is consistent with the lipase being in the native open conformation at the water/hydrophobic interface.