Insight into heterogeneity in cell-surface hydrophobicity and ability to degrade hydrocarbons among cells of two hydrocarbon-degrading bacterial populations

The sequential bacterial adherence to hydrocarbons (BATH) of successive generations of hydrophobic fractions of Paenibacillus sp. R0032A and Burkholderia cepacia gave rise to bacterial populations of increasing cell-surface hydrophobicity. Thus, hydrophobicity of the first generation (H1) was less than that of the second generation (H2), which was less than that of the third generation (H3). Beyond H3, the hydrophobic populations became less stable and tended to lyse in hexadecane after violent (vortex) agitation, resulting in an apparent decline in BATH value. The exhaustively fractionated aqueous-phase population (L) was very hydrophilic. The overall cell-surface distribution of the population was L < parental strain < H1 < H2 < H3. The ability to degrade crude oil, hexadecane, or phenanthrene matched the degree of cell-surface hydrophobicity: L < P < H1 < H2 < H3. Thus, in natural populations of hydrocarbon-degrading Paenibacillus sp. R0032A and B. cepacia, there is a heterogeneity in the hydrophobic surface characteriistics that affects the ability of cells to use various hydrocarbon substrates.     

Determination of the cell surface hydrophobicity of oral bacteria using a modified hydrocarbon adherence method

Abstract Hydrophobic interactions between bacterial cell surfaces and colonisable substrates have been implicated in the mechanisms of bacterial adherence. However, current methods of assessing bacterial hydrophobicity as a function of adherence to liquid hydrocarbons (especially hexadecane) do not always produce accurate or reproducible results. Therefore, the present technique was developed using xylene. The hydrophobic surface properties of fresh and type strains of Bacteriodes gingivalis, Bacteriodes intermedius, Capnocytophaga spp., Streptococcus salivarius and Streptococcus sanguis suspended either in saliva ions buffer (SIB) or in saliva diluted in SIB were measured. In SIB the test strains were predominantly hydrophobic. The addition of saliva caused a significant reduction ( P < 0.05) in hydrophobicity compared to SIB alone, with 80% of the strains tested. Since oral bacteria will be suspended in saliva in vivo, it is concluded that bacteria in the oral cavity may be less hydrophobic than previous studies have suggested.     

Standardization of salt aggregation test for reproducible determination of cell-surface hydrophobicity with special reference to Staphylococcus species

The laboratory conditions for reproducible routine determination of staphylococcal cell-surface hydrophobicity by the salt aggregation test were standardized. Fresh bacterial suspensions standardized to 5 x 10(9) cfu/ml gave the most reproducible results with both Staphylococcus aureus and coagulase-negative staphylococci. For relatively hydrophobic strains a 5-min reading time was necessary to detect bacterial aggregation in ammonium sulphate solutions ranging from 0.1 M to 1.5 M, pH 6.8. A x 10 hand lens facilitated reading aggregations. Overnight storage of bacterial suspensions at 20 degrees C reduced cell-surface hydrophobicity of all species, while storage at 4 degrees C reduced the hydrophobic nature of Staph. aureus strains. The hydrophobicity of coagulase-negative staphylococci rarely changed at 4 degrees C. A 10-fold dilution of fresh, standardized bacterial suspensions made it impossible to detect bacterial aggregation in ammonium sulphate solutions even with a hand lens. Under standardized conditions three types of staphylococcal cell aggregations were observed. The first looked like the slide agglutination for O antigens of Enterobacteriaceae, the second resembled H-agglutination, while the third had a filamentous appearance. These patterns indicated that more than one component might contribute to cell-surface hydrophobicity of both Staph. aureus and coagulase-negative staphylococci, or the same component might have different position on the cell surface.     

Standardization of salt aggregation test for reproducible determination of cell-surface hydrophobicity with special reference to Staphylococcus species

The laboratory conditions for reproducible routine determination of staphylococcal cell-surface hydrophobicity by the salt aggregation test were standardized. Fresh bacterial suspensions standardized to 5 times 10⁹ cfu/ml gave the most reproducible results with both Staphylococcus aureus and coagulase-negative staphylococci. For relatively hydrophobic strains a 5-min reading time was necessary to detect bacterial aggregation in ammonium sulphate solutions ranging from 0.1 M to 1.5 M, pH 6.8. A × 10 hand lens facilitated reading aggregations. Overnight storage of bacterial suspensions at 20C reduced cell-surface hydrophobicity of all species, while storage at 4C reduced the hydrophobic nature of Staph. aureus strains. The hydrophobicity of coagulase-negative staphylococci rarely changed at 4C. A 10-fold dilution of fresh, standardized bacterial suspensions made it impossible to detect bacterial aggregation in ammonium sulphate solutions even with a hand lens. Under standardized conditions three types of staphylococcal cell aggregations were observed. The first looked like the slide agglutination for O antigens of Enterobacteriaceae, the second resembled H-agglutination, while the third had a filamentous appearance. These patterns indicated that more than one component might contribute to cell-surface hydrophobicity of both Staph. aureus and coagulase-negative staphylococci, or the same component might have different position on the cell surface.     

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.     

Effect of R-plasmid RP1 on surface hydrophobicity of Proteus mirabilis

The presence of R-plasmid RP1, as well as the conditions of growth, affected the surface hydrophobicity of a clinical isolate of Proteus mirabilis. However, results depended upon the method of assessment. Stationary phase plasmid-containing cells appeared to be less hydrophobic than plasmid-free cells when hydrophobicity was measured by the contact angle method, but more hydrophobic when measured by bacterial adherence to hydrocarbons or hydrophobic interaction chromatography. Cells growing in a chemostat differed in hydrophobicity from stationary phase cells and results varied with the growth rate. Plasmid-mediated effects were greatest in iron-depleted cells, and differences between plasmid-containing and plasmid-free cells were virtually eliminated by pre-treatment with antiserum.     

Putative role of a 70 kDa outer-surface protein in promoting cell-surface hydrophobicity of Serratia marcescens RZ

Serratia marcescens RZ has been previously shown to possess pronounced cell-surface hydrophobicity, as evidenced by its affinity for hydrocarbons and polystyrene. The present report suggests the involvement of a 70 kDa protein, serraphobin, in this phenomenon. The 70 kDa protein was recovered from both the cell surface and culture supernatant of hydrophobic wild-type cells, but was either totally absent or present in minor quantities in hydrophobicity-deficient mutants. Similarly, loss of hydrophobicity of RZ cells following growth at 39 degrees C was accompanied by loss of the protein. Serraphobin was capable of binding to hexadecane droplets following a brief mixing procedure, and could be desorbed by solidifying and melting the hexadecane phase.     

Isolation of pigmented and nonpigmented mutants of Serratia marcescens with reduced cell surface hydrophobicity

Enrichment for nonhydrophobic mutants of Serratia marcescens yielded two types: (i) a nonpigmented mutant which exhibited partial hydrophobic characteristics compared with the wild type, as determined by adherence to hexadecane and polystyrene; and (ii) a pigmented, nonhydrophobic mutant whose colonies were translucent with respect to those of the wild type. The data suggest that the pronounced cell surface hydrophobicity of the wild type is mediated by a combination of several surface factors.     

Effect of a Pseudomonas rhamnolipid biosurfactant on cell hydrophobicity and biodegradation of octadecane.

In this study, the effect of a purified rhamnolipid biosurfactant on the hydrophobicity of octadecane-degrading cells was investigated to determine whether differences in rates of octadecane biodegradation resulting from the addition of rhamnolipid to four strains of Pseudomonas aeruginosa could be related to measured differences in hydrophobicity. Cell hydrophobicity was determined by a modified bacterial adherence to hydrocarbon (BATH) assay. Bacterial adherence to hydrocarbon quantitates the preference of cell surfaces for the aqueous phase or the aqueous-hexadecane interface in a two-phase system of water and hexadecane. On the basis of octadecane biodegradation in the absence of rhamnolipid, the four bacterial strains were divided into two groups: the fast degraders (ATCC 15442 and ATCC 27853), which had high cell hydrophobicities (74 and 55% adherence to hexadecane, respectively), and the slow degraders (ATCC 9027 and NRRL 3198), which had low cell hydrophobicities (27 and 40%, respectively). Although in all cases rhamnolipid increased the aqueous dispersion of octadecane at least 10(4)-fold, at low rhamnolipid concentrations (0.6 mM), biodegradation by all four strains was initially inhibited for at least 100 h relative to controls. At high rhamnolipid concentrations (6 mM), biodegradation by the fast degraders was slightly inhibited relative to controls, but the biodegradation by the slow degraders was enhanced relative to controls. Measurement of cell hydrophobicity showed that rhamnolipids increased the cell hydrophobicity of the slow degraders but had no effect on the cell hydrophobicity of the fast degraders. The rate at which the cells became hydrophobic was found to depend on the rhamnolipid concentration and was directly related to the rate of octadecane biodegradation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Physico-chemical surface characteristics and adhesive properties of Streptococcus salivarius strains with defined cell surface structures

Physico-chemical surface characteristics and adhesive properties of a series of mutants of Streptococcus salivarius HB with defined cell surface structures were determined. Zeta potentials showed no relation either with the presence or absence of specific antigens on the bacterial cell surface, or with the adhesive properties of the cells. Hydrophobicity was assessed by surface free energy determination from measured contact angles, by adsorption to hexadecane and by hydrophobic interaction chromatography. Generally, the progressive removal of fibril subclasses from the cell surface resulted in a reduced hydrophobicity. However, specific fibrillar subclasses appeared to contribute to surface hydrophobicity to widely different extents. Bacterial adhesion to polymethylmethacrylate increased with increasing hydrophobicity of the mutants. However, adhesion to a more complex biological substratum, such as saliva-coated hydroxyapatite, correlated only partly with hydrophobicity. The organism, deprived of most of its fibrillar surface structures, clearly showed the least adhesion to hydrophobic ligands, to both polymethylmethacrylate and saliva-coated hydroxyapatite, and had a significantly higher surface free energy than the other mutants and the parent strain.     

Influence of the growth conditions on the hydrophobicity of Renibacterium salmoninarum evaluated by different methods

The influence of medium and salinity on the cell surface hydrophobicity of Renibacterium salmoninarum was investigated using three different methods: bacterial adherence to hydrocarbons (BATH), salt agglutination test (SAT), and binding to nitrocellulose filters (NCF). The possible relationship among hydrophobicity, haemagglutination and adherence to cell lines was also evaluated. R. salmoninarum showed to be highly hydrophobic regardless of the growth conditions or technique employed. Nevertheless, slight differences can be detected depending on the method used. In the SAT and NCF assays very uniform values were obtained within the strains. All the R. salmoninarum isolates agglutinated in (NH4)2SO4 in a range of 0.05-0.2 M and displayed a 77-100% of adherence to nitrocellulose filters. However, more variable results were observed in the BATH method depending on the hydrocarbon, buffer and strain employed. Although all of the isolates produced haemagglutinins for homeotherm erythrocytes, the majority of them failed to agglutinate poikilothermic red blood cells and were unable to adhere to fish cell lines. Therefore, a general correlation among hydrophobicity, agglutinating capacity for fish erythrocytes and adherence to fish cells can not be established for R. salmoninarum.     

Hydrophobicity of Bacillus and Clostridium spores.

The hydrophobicities of spores and vegetative cells of several species of the genera Bacillus and Clostridium were measured by using the bacterial adherence to hexadecane assay and hydrophobic interaction chromatography. Although spore hydrophobicity varied among species and strains, the spores of each organism were more hydrophobic than the vegetative cells. The relative hydrophobicities determined by the two methods generally agreed. Sporulation media and conditions appeared to have little effect on spore hydrophobicity. However, exposure of spore suspensions to heat treatment caused a considerable increase in spore hydrophobicity. The hydrophobic nature of Bacillus and Clostridium spores suggests that hydrophobic interactions may play a role in the adhesion of these spores to surfaces.     

Hydrophobicity of Bacillus and Clostridium spores

The hydrophobicities of spores and vegetative cells of several species of the genera Bacillus and Clostridium were measured by using the bacterial adherence to hexadecane assay and hydrophobic interaction chromatography. Although spore hydrophobicity varied among species and strains, the spores of each organism were more hydrophobic than the vegetative cells. The relative hydrophobicities determined by the two methods generally agreed. Sporulation media and conditions appeared to have little effect on spore hydrophobicity. However, exposure of spore suspensions to heat treatment caused a considerable increase in spore hydrophobicity. The hydrophobic nature of Bacillus and Clostridium spores suggests that hydrophobic interactions may play a role in the adhesion of these spores to surfaces.     

Biosurfactants,an help in the biodegradation of hexadecane? The case of <Emphasis Type="Italic">Rhodococcus</Emphasis> and <Emphasis Type="Italic">Pseudomonas</Emphasis> strains

The roles of the extracellular biosurfactants produced by two bacterial strains, Pseudomonas aeruginosa GL1 and Rhodococcus equi Ou2, in hexadecane uptake and biodegradation were compared. For this purpose, cell hydrophobicity and production of glycolipidic biosurfactants were evaluated during bacterial growth on hexadecane, as well the effects of these biosurfactants on culture supernatants properties i.e., surface and interfacial tensions, and emulsification and pseudosolubilization capacities. The results showed that the role of biosurfactants was different in these two strains and was directly related to the hydrophobicity of the bacterial cells concerned. Extracellular biosurfactants produced by strain R. equi Ou2 had only a minor role in hexadecane degradation. Direct interfacial accession appeared to be the main mechanism for hexadecane uptake by the hydrophobic cells of strain R. equi Ou2. On the contrary, the biosurfactants produced by P. aeruginosa GL1 were required for growth on hexadecane, and their pseudosolubilization capacity rather than their emulsification capacity was involved in substrate degradation, allowing uptake from hexadecane micelles by the hydrophilic cells of this bacterium. The roles of biosurfactants thus differ widely among bacteria degrading hydrophobic compounds. J.-P. Vandecasteele—in retirement     

Effect of aminoglycosides on surface hydrophobicity of Acinetobacter baumannii

Effects of amikacin, gentamicin, netilmicin and tobramycin at subinhibitory concentrations (sub-MICs) (11/4, 1/8, 1/16 or 1/32 of their MICs) on the cell surface hydrophobicity of two Acinetobacter baumannii strains (7194 and 16265) were evaluated. Hydrophobicity was determined by two different methods - by adherence of bacteria to hydrocarbon (xylene) and by aggregation of bacteria in ammonium sulphate solutions at various concentrations. The adherence of A. baumannii strains to xylene decreased, mainly, after treatment with netilmicin at 1/4, 1/8 or 1/16 of the MIC (to 6.4%, 17.0% or 24.5% of the control value) (strain 7194) and after treatment with amikacin and gentamicin at 1/4 of their MICs (to 58.4% or 54.4%) (strain 16265). A decrease in surface hydrophobicity of exposed strains under these conditions was shown in salting-out test, too. Tobramycin reduced hydrophobic properties of A. baumannii strains at all tested sub-MICs to only a small extent.     

High surface hydrophobicity of hemagglutinatingVibrio cholerae and other vibrios

Surface hydrophobicity of hemagglutinatingVibrio cholerae, Vibrio parahaemolyticus, and NAG vibrios has been investigated. Most strains caused mannose-sensitive hemagglutination of monkey, guinea pig, chicken, and mannose-resistant hemagglutination of human erythrocytes with different degrees of hemagglutinating activity. Hemagglutinating strains adsorbed to a hydrophobic gel (Octyl Sepharose), whereas nonhemagglutinating strains failed to adsorb.Vibrio cholerae and other vibrios investigated seem to have pronounced surface hydrophobicity as estimated by Octyl Sepharose and they correspondingly autoaggregated into visible cell clumps in ammonium sulfate solution at low molarity (0.2–0.4 M). Nonhemagglutinating strains did not aggregate even at high (2 M) ammonium sulfate concentration. The presence of surface hemagglutinins of vibrios is growth-media-dependent. Strains, grown in four different liquid media, produced hemagglutinins and expressed pronounced surface hydrophobicity. Studies with electron microscopy revealed the presence of fimbriae on the vibrio cells. The number of fimbriae on the cells varied from strain to strain. Some strains possessed more than 300 fimbriae/cell whereas others had less than 10 fimbriae/cell. Vibrio hemagglutinins are easily detached from the cell surface by heating or sonication, and their cell surface hydrophobicity decreased simultaneously.     

Characterization of bacterial isolates from industrial wastewater according to probable modes of hexadecane uptake

Bacterial isolates from industrial wastewater were characterized according to probable modes of hexadecane uptake based on data for cell surface hydrophobicity, emulsifying activity, glycoside content and surface tension of cell-free culture medium. The results obtained suggested that both modes of biosurfactant-enhanced hexadecane uptake by bacterial strains take place, direct uptake and alkane transfer. The increase in cell surface hydrophobicity and glycoside production by the strains suggested the existence of biosurfactant-enhanced interfacial uptake of the alkane. Such mechanism is probably predominant for three isolates, Staphylococcus sp. HW-2, Streptococcus sp. HW-9 and Bacillus sp. HW-4. Secreted biosurfactants enhanced mainly alkane emulsification for most hydrophobic isolate Arthrobacter sp. HW-8, and micellar transfer for most hydrophilic isolate Streptococcus sp. HW-5. For other strains (67%) both mechanisms of biosurfactant-enhanced hexadecane uptake probably take place in similar degree, interfacial uptake and alkane emulsification. The results obtained could contribute to clarifying the natural relationships between the members of water ecosystem studied as well as will reveal potential producers of surface active compounds.     

Hydrophobicity, Adhesion, and Surface-Exposed Proteins of Gliding Bacteria

The cell surface hydrophobicities of a variety of aquatic and terrestrial gliding bacteria were measured by an assay of bacterial adherence to hydrocarbons (BATH), hydrophobic interaction chromatography, and the salt aggregation test. The bacteria demonstrated a broad range of hydrophobicities. Results among the three hydrophobicity assays performed on very hydrophilic strains were quite consistent. Bacterial adhesion to glass did not correlate with any particular measure of surface hydrophobicity. Several adhesion-defective mutants of Cytophaga sp. strain U67 were found to be more hydrophilic than the wild type, particularly by the BATH assay and hydrophobic interaction chromatography. The very limited adhesion of these mutants correlated well with hydrophilicity as determined by the BATH assay. The hydrophobicities of several adhesion-competent revertants ranged between those of the wild type and the mutants. As measured by the BATH assay, starvation increased hydrophobicity of both the wild type and an adhesion-defective mutant. During filament fragmentation of Flexibacter sp. strain FS-1, marked changes in hydrophobicity and adhesion were accompanied by changes in the arrays of surface-exposed proteins as detected by an immobilized radioiodination procedure.     

Sequential hydrophobic partitioning of cells of Pseudomonas aeruginosa gives rise to variants of increasing cell-surface hydrophobicity

The partitioning of bacterial cells in a dual aqueous-solvent phase system leads to separation into 'hydrophilic' and hydrophobic functions. Sequential multistep partitioning, accompanied by successive enrichment, gives rise to several cycles of hydrophobic and hydrophilic cell populations which possess different cell-surface hydrophobicity characteristics. Characterization of the cell-surface hydrophobicity by several methods (salting-out aggregation test, bacterial adherence to hydrocarbon, polystyrene binding and hydrophobic interaction chromatography) was carried out. The cell-surface hydrophobicity varied in the order: hydrophilic fraction < parental strain < first cycle hydrophobic variant < second cycle hydrophobic variant < third cycle hydrophobic variant. Electron microscopy showed that the most hydrophobic variant was densely covered by hydrophobic structures - fimbriae - whereas the parental strain was covered by a mixture of surface structures. The hydrophilic variant was covered by an amorphous exopolymeric substance, which is a polysaccharide, shown by its reaction with Alcian blue.