Adsorption behavior and enzymatically or chemically induced cross‐linking of a mussel adhesive protein

The adsorption behavior of the mussel adhesive protein Mytilus edulis foot protein‐1 (Mefp‐1) has been investigated on a negatively charged polar SiO₂ surface and an electrically inert non‐polar CH₃‐terminated thiolated gold surface. How the structure of adsorbed Mefp‐1 is changed upon chemically and enzymatically induced cross‐linking using sodium periodate (NaIO₄) and catechol oxidase, both of which transform DOPA residues in Mefp‐1 into highly reactive o‐quinones, was also investigated. The results are compared with those resulting from addition of Cu²⁺ to adsorbed Mefp‐1, which forms complexes with and catalyses oxidation of DOPA residues, previously suggested to participate in the cohesive and adhesive properties of the byssus thread of M. edulis. By combining surface plasmon resonance (SPR) and quartz crystal microbalance/dissipation (QCM‐D) measurements, the effects of these agents were investigated with respect to changes in the amount of coupled water, the viscoelastic properties (rigidity) and the hydrodynamic thickness of the protein adlayers. The layer of Mefp‐1 formed on the bare CH₃‐terminated surface was elongated, flexible and coupled hydrodynamically a substantial amount of water, whereas Mefp‐1 formed a rigidly attached adlayer on the SiO₂ surface. Upon enzymatically and chemically induced cross‐linking of Mefp‐1 formed on the CH₃ surface, the rigidity of the adlayer(s) increased significantly. A similar increase in the rigidity was observed also upon addition of Cu²⁺, suggesting that the high level of metal ions present in the byssus thread might be essential for the cohesive and adhesive properties of this protein. For the mass‐uptake kinetics of enzymatically induced cross‐linking, three different phases were observed and are interpreted as competition between binding of protein and release of coupled water. For the reaction with NaIO₄ and Cu²⁺, only release of water affected the coupled mass. The importance of this type of information for an improved understanding of the strong adhesion and cohesive properties in marine environments is discussed.     

Polyphosphoprotein from the adhesive pads of Mytilus edulis

Achieving a satisfactory biochemical explanation for the opportunistic underwater adhesion of marine invertebrates such as mussels and barnacles requires a detailed characterization of proteins extracted from holdfast structures produced by these organisms. Mefp-5 is an adhesive protein derived from the foot of the common mussel, Mytilus edulis, and deposited into the byssal attachment pads. Purification and primary structure of mefp-5 was determined by peptide mapping and cDNA sequencing. The protein is 74 residues long and has a mass of about 9500 Da. Mefp-5 composition shows a strong amino acid bias: aromatic amino acids, lysine, and glycine represent 65 mol % of the composition. More than a third of all the residues in the protein are posttranslationally modified by hydroxylation or phosphorylation. The conversion of tyrosine to 3, 4-dihydroxyphenyl-L-alanine (DOPA) and serine to O-phosphoserine accounts for the hydroxylation and phosphorylation, respectively. Neither modification is complete since variations in the extent of phosphorylation and hydroxylation can be detected by mass spectrometry. More than 75% of the DOPA is adjacent to basic residues, e.g., Lys-DOPA and DOPA-Lys. Phosphoserine occurs in sequences strikingly reminiscent of acidic mineral-binding motifs that appear in statherin, osteopontin, and others. This may be an adaptation for adhesion to the most common substrata for mussels, i.e., calcareous materials.     

Cell surface properties as factors involved in Proteus vulgaris adhesion to stainless steel under starvation conditions

The purpose of these investigations was to evaluate the influence of limited nutrient availability in the culture medium on Proteus vulgaris biofilm formation on surfaces of stainless steel. The relationship between the P. vulgaris adhesion to the abiotic surfaces, the cellular ATP levels, cell surface hydrophobicity and changes in the profiles of extracellular proteins and lipopolysaccharides was examined. In all experimental variants the starvation conditions induced the bacterial cells to adhere to the surfaces of stainless steel. Higher ATP content and lower cell surface hydrophobicity of P. vulgaris cells was observed upon nutrient-limited conditions. Under starvation conditions a reduction in the levels of extracellular low molecular weight proteins was noticed. High molecular weight proteins formed the conditioning layer on stainless steel plates, making the bacteria adhesion process more favorable. The production of low molecular weight carbohydrates promoted more advanced stages of P. vulgaris biofilm formation process on the surfaces of stainless steel upon starvation.     

Evaluation of Bacillus anthracis and Yersinia pestis sample collection from nonporous surfaces by quantitative real‐time PCR

Aim: We will validate sample collection methods for recovery of microbial evidence in the event of accidental or intentional release of biological agents into the environment. Methods and Results: We evaluated the sample recovery efficiencies of two collection methods – swabs and wipes – for both nonvirulent and virulent strains of Bacillus anthracis and Yersinia pestis from four types of nonporous surfaces: two hydrophilic surfaces, stainless steel and glass, and two hydrophobic surfaces, vinyl and plastic. Sample recovery was quantified using real‐time qPCR to assay for intact DNA signatures. We found no consistent difference in collection efficiency between swabs or wipes. Furthermore, collection efficiency was more surface‐dependent for virulent strains than nonvirulent strains. For the two nonvirulent strains, collection efficiency was similar between all four surfaces, albeit B. anthracis Sterne exhibited higher levels of recovery compared to Y. pestis A1122. In contrast, recovery of B. anthracis Ames spores and Y. pestis CO92 from the hydrophilic glass or stainless steel surfaces was generally more efficient compared to collection from the hydrophobic vinyl and plastic surfaces. Conclusions: Our results suggest that surface hydrophobicity may play a role in the strength of pathogen adhesion. The surface‐dependent collection efficiencies observed with the virulent strains may arise from strain‐specific expression of capsular material or other cell surface receptors that alter cell adhesion to specific surfaces. Significance and Impact of the Study: These findings contribute to the validation of standard bioforensics procedures and emphasize the importance of specific strain and surface interactions in pathogen detection.     

Optimization of l‐DOPA production by Brevundimonas sp. SGJ using response surface methodology

l ‐DOPA (3,4‐dihydroxyphenyl‐l ‐alanine) is an extensively used drug for the treatment of Parkinson's disease. In the present study, optimization of nutritional parameters influencing l ‐DOPA production was attempted using the response surface methodology (RSM) from Brevundimonas sp. SGJ. A Plackett–Burman design was used for screening of critical components, while further optimization was carried out using the Box–Behnken design. The optimized levels of factors predicted by the model were pH 5.02, 1.549 g l^?1 tryptone, 4.207 g l^?1 l ‐tyrosine and 0.0369 g l^?1 CuSO₄, which resulted in highest l ‐DOPA yield of 3.359 g l^?1. The optimization of medium using RSM resulted in a 8.355‐fold increase in the yield of l ‐DOPA. The anova showed a significant R² value (0.9667), model F‐value (29.068) and probability (0.001), with insignificant lack of fit. The highest tyrosinase activity observed was 2471 U mg^?1 at the 18th hour of the incubation period with dry cell weight of 0.711 g l^?1. l ‐DOPA production was confirmed by HPTLC, HPLC and GC‐MS analysis. Thus, Brevundimonas sp. SGJ has the potential to be a new source for the production of l ‐DOPA.     

Adsorption of biosurfactant on solid surfaces and consequences regarding the bioadhesion of Listeria monocytogenes LO28

AIMS: The influence of biosurfactant compounds produced by a strain of Pseudomonas fluorescens on the adhesion of Listeria monocytogenes LO28 to polytetrafluoroethylene (PTFE) and AISI 304 stainless steel surfaces was investigated. METHODS AND RESULTS: The biosurfactant was produced according to a simple, novel technique based on cultivation on nutrient agar. Adhesion studies were performed using L. monocytogenes cells cultured at 20 or 37 degrees C. CONCLUSIONS: A substrate-dependent behaviour of the LO28 strain (larger number of cells adhering to stainless steel than to PTFE), and a significant reduction (< 90%) in microbial adhesion levels through the prior adsorption of biosurfactants on stainless steel surfaces, which can be related to a change in the electron-donor characteristics of this substratum, was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The prior adsorption of biosurfactants on solid surfaces may constitute a new and effective means of combating the implantation of pathogenic micro-organisms in food processing plants.     

The effect of end‐group substitution on surface self‐assembly of peptides

Biofouling, the undesirable accumulation of organisms onto surfaces, affects many areas including health, water, and energy. We previously designed a tripeptide that self‐assembles into a coating that prevents biofouling. The peptide comprises three amino acids: DOPA, which allows its adhesion to the surface, and two fluorinated phenylalanine residues that direct its self‐assembly into a coating and acquire it with antifouling properties. This short peptide has an ester group at its C‐terminus. To examine the importance of this end group for the self‐assembly and antifouling properties of the peptide, we synthesized and characterized tripeptides with different end groups (ester, amide, or carboxylic group). Our results indicate that different groups at the C‐terminus of the peptide can lead to a change in the peptide assembly on the surface and its adsorption process. However, this change only affects the antifouling properties of the coating toward Gram‐positive bacteria (Staphylococcus epidermidis), whereas Gram‐negative bacteria (Escherichia coli) are not affected.     

Role of hydrophobicity in adhesion of wild yeast isolated from the ultrafiltration membranes of an apple juice processing plant

The role of cell surface hydrophobicity in the adhesion to stainless steel (SS) of 11 wild yeast strains isolated from the ultrafiltration membranes of an apple juice processing plant was investigated. The isolated yeasts belonged to four species: Candida krusei (5 isolates), Candida tropicalis (2 isolates), Kluyveromyces marxianus (3 isolates) and Rhodotorula mucilaginosa (1 isolate). Surface hydrophobicity was measured by the microbial adhesion to solvents method. Yeast cells and surfaces were incubated in apple juice and temporal measurements of the numbers of adherent cells were made. Ten isolates showed moderate to high hydrophobicity and 1 strain was hydrophilic. The hydrophobicity expressed by the yeast surfaces correlated positively with the rate of adhesion of each strain. These results indicated that cell surface hydrophobicity governs the initial attachment of the studied yeast strains to SS surfaces common to apple juice processing plants.     

Antioxidant efficacy and adhesion rescue by a recombinant mussel foot protein‐6

Mytilus foot protein type 6 (mfp‐6) is crucial for maintaining the reducing conditions needed for optimal wet adhesion in marine mussels. In this report, we describe the expression and production of a recombinant Mytilus californianus foot protein type 6 variant 1 (rmfp‐6.1) fused with a hexahistidine affinity tag in Escherichia coli and its purification by affinity chromatography. Recombinant mfp‐6 showed high purification yields of 5–6 mg L^?1 cell culture and excellent solubility in low pH buffers that retard oxidation of its many thiol groups. Purified rmfp‐6.1 protein showed high 2,2‐diphenyl‐1‐picrylhydrazyl radical scavenging activity when compared with vitamin C. Using the highly sensitive surface forces apparatus (SFA) technique to measure interfacial surface forces in the nano‐Newton range, we show that rmfp‐6.1 is also able to rescue the oxidation‐dependent adhesion loss of mussel foot protein 3 (mfp‐3) at pH 3. The adhesion rescue is related to a reduction of dopaquinone back to 3,4‐dihydroxyphenyl‐l ‐alanine in mfp‐3, which is the reverse reaction observed during the detrimental enzymatic browning process in fruits and vegetables. Broadly viewed, rmfp‐6.1 has potential as a versatile antioxidant for applications ranging from personal products to antispoilants for perishable foods during processing and storage. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1587–1593, 2013     

Surfactin reduces the adhesion of food-borne pathogenic bacteria to solid surfaces

Aims:  To investigate the effect of the biosurfactants surfactin and rhamnolipids on the adhesion of the food pathogens Listeria monocytogenes , Enterobacter sakazakii and Salmonella Enteritidis to stainless steel and polypropylene surfaces.
Methods and Results:  Quantification of bacterial adhesion was performed using the crystal violet staining technique. Preconditioning of surfaces with surfactin caused a reduction on the number of adhered cells of Ent. sakazakii and L. monocytogenes on stainless steel. The most significant result was obtained with L. monocytogenes where number of adhered cells was reduced by 10² CFU cm⁻². On polypropylene, surfactin showed a significant decrease on the adhesion of all strains. The adsorption of surfactin on polystyrene also reduces the adhesion of L. monocytogenes and Salm. Enteritidis growing cells. For short contact periods using nongrowing cells or longer contact periods with growing cells, surfactin was able to delay bacterial adhesion.
Conclusions:  The prior adsorption of surfactin to solid surfaces contributes on reducing colonization of the pathogenic bacteria.
Significance and Impact of the Study:  This is the first work investigating the effect of surfactin on the adhesion of the food pathogens L. monocytogenes , Ent. sakazakii and Salm. Enteritidis to polypropylene and stainless steel surfaces.     

Effects of double bands on Magellanic Penguins

ABSTRACT Banding penguins is controversial because bands can alter the survival, reproduction, and behavior of marked individuals. The effects of bands are not consistent among band types and, although stainless steel is thought to be better than other materials, tests of the long‐term impact of bands on tag‐loss rates and the reproduction and survival of individuals are needed. We tested three types of external tags on Magellanic Penguins (Spheniscus magellanicus) to measure band effects and tag‐loss rates. In 1993, we double‐tagged 300 penguins with aluminum flipper bands, stainless‐steel flipper bands, or small (2 mm × 10 mm) metal tags attached to foot webbing. We searched for double‐tagged birds for 13 of 15 yrs (1994–2008). Aluminum bands deformed, caused feather wear, injured and killed some penguins, and were lost more often than stainless‐steel bands or web tags. During the first 2 yrs of our study, at least nine penguins lost one aluminum band (N= 71 penguins resighted), but no penguins lost a stainless‐steel band (N= 84) or a web tag (N= 88). During the next 13 yrs, five penguins lost one of their two web tags (N= 89), but none lost a stainless‐steel band (N= 84). Females laid eggs of similar size before they carried a band and in the year following tagging (P= 0.09). The type of tags a female carried did not significantly change egg size (P > 0.22). During the first breeding season after tagging, penguins with aluminum bands had lower reproductive success than penguins with stainless‐steel bands or web‐tags (P= 0.04). The annual survival of females with two stainless‐steel bands was lower (0.79) than that of males with two stainless‐steel bands or males and females with two web‐tags (0.87). Aluminum bands injured Magellanic Penguins, were lost at high rates, and should not be used. Double stainless‐steel bands had no apparent effects on adult male Magellanic Penguins, but reduced survival rates of adult females. A single stainless‐steel band would likely have less impact than two bands, and our results suggest that the impact of a single band would be difficult to measure.     

Multiple drug resistance and strength of attachment to surfaces in Pseudomonas aeruginosa isolates

Aims: To investigate the presence of a relationship between the strength of attachment of Pseudomonas aeruginosa to stainless steel surfaces and their observed multiple drug resistance. Methods and Results: Multiple drug resistance of clinical and environmental isolates of Ps. aeruginosa was evaluated using disc diffusion method. The blot succession technique was used to quantify the strength of attachment of Ps. aeruginosa isolates. Different multiple drug–resistant Ps. aeruginosa isolates exhibited variable attachment strength. Although the highest multiple drug–resistant clinical isolate was shown to have the least attachment strength among clinical isolates, a weak correlation was found between attachment strength and multiple resistance among our investigated Ps. aeruginosa isolates. Conclusions: There is a weak correlation between multiple drug resistance and strength of attachment to stainless steel surfaces. Significance and Impact of the Study: Even low‐resistant Ps. aeruginosa could have the potential of attaching firmly to surfaces and forming biofilm.     

Growth phase-dependent surface properties of Legionella pneumophila and their role in adhesion to stainless steel coated QCM-D sensors

Legionella pneumophila cell surface hydrophobicity and charge are important determinants of their mobility and persistence in engineered water systems (EWS). These surface properties may differ depending on the growth phase of L. pneumophila resulting in variable adhesion and persistence within EWS. We describe the growth-dependent variations in L. pneumophila cell surface hydrophobicity and surface charge using the microbial adhesion to hydrocarbon assay and microelectrophoresis, respectively, and their role in cell adhesion to stainless steel using a quartz crystal microbalance with dissipation (QCM-D) monitoring instrument. We observed a steady increase in L. pneumophila hydrophobicity during their lifecycle in culture media. Cell surfaces of stationary phase L. pneumophila were significantly more hydrophobic than their lag and midexponential counterparts. No significant changes in L. pneumophila cell surface charge were noted. Morphology of L. pneumophila remained relatively constant throughout their lifecycle. In the QCM-D study, lag and exponential phase L. pneumophila weakly adhered to stainless steel surfaces resulting in viscoelastic layers. In contrast, stationary phase bacteria were tightly and irreversibly bound to the surfaces, forming rigid layers. Our results suggest that the stationary phase of L. pneumophila would highly favour their adhesion to plumbing surfaces and persistence in EWS.     

Adsorption of flavobacterium breve and pseudomonas fluorescens p17 on different metals: Electrochemical polarization effect

The electrochemical polarization effect on early adsorption of Flavobacterium breve and Pseudomonas fluorescens P17 to platinum, titanium, stainless steel, copper, aluminum alloy and mild steel was studied. A well‐defined peak characterized the bacterial adsorption dependence on externally applied potential. Maximal adsorption occurred in the potential range of ‐0.5 to 0.5 V (SCE) for all tested metals. A shift of applied potential towards both a positive and a negative direction from the maximal adsorption potential (Emax,ad) was accompanied by a gradual decrease in bacterial adsorption. The extent of bacterial adsorption strongly depended on the nature of the metallic substratum and decreased accordingly as follows: platinum > titanium > stainless steel > aluminum alloy > carbon steel > copper. Adsorption on all tested metals was approximately two orders of magnitude higher with the relatively more hydrophobic F. breve compared to the less hydrophobic P. fluorescens P17. The effect of electrochemical polarization on the initial stages of bacterial adsorption onto metallic substrata is further discussed.     

Removal kinetics of Bacillus cereus spores from a stainless steel surface exposed to constant shear stress 1 ‐ experimental system

The efficiency of cleaning in place procedures in dairy industries can be greatly affected by the presence of spore‐forming bacteria, which are able to adhere strongly to surfaces and to survive disinfection procedures. Microbial adhesion has been extensively studied, but very few studies have yet reported on the hydrodynamic removal of microorganisms, due to the lack of simple, routinely performable techniques. In this paper, a methodology using a coaxial cylinder double gap viscometer is described, to study the removal kinetics of Bacillus cereus spores from a stainless steel support under hydrodynamic conditions. This method was shown to be reproducible, sensitive and easy to perform, and allowed spore hydrodynamic removal kinetics to be studied as a function of both adhesion and detachment conditions. A high ionic strength attachment medium was shown to enhance adhesion forces, provided it did not contain macromolecules. An increase in shear stress was found to be favorable to spore detachment (4 to 5 times more spores were detached at 28 Pa than at 2 Pa), but removal kinetics were not found to be significantly different for 2 and 15 Pa. Thus, the effect of shear stress on spore removal kinetics may not be linear.     

CagA tyrosine phosphorylation in gastric epithelial cells caused by Helicobacter pylori in patients with gastric adenocarcinoma

Background. Tyrosine phosphorylation of Helicobacter pylori cytotoxin‐associated protein of in gastric epithelial cells is reported. The goals of this study are first to examine the occurrence of CagA tyrosine phosphorylation in H. pylori strains isolated from patients with gastric adenocarcinoma and gastritis, and second to clarify the relationship between the diversity of tyrosine phosphorylation motifs and the presence of CagA tyrosine phosphorylation. Methods. Fifty‐eight clinical isolates of H. pylori from patients with gastric adenocarcinoma (29 cases) and gastritis (29 cases) were studied for CagA tyrosine phosphorylation by Western blotting. Sequence diversity of tyrosine phosphorylation motifs was analysed among positive‐ or negative‐CagA tyrosine phosphorylation isolates. Results. Positive CagA tyrosine phosphorylation was found in 93.1% (27 of 29) of strains from gastric adenocarcinoma patients and 51.7% (15 of 29) of strains from gastritis patients (p < 0.001). Intact motifs were found in H. pylori isolates with CagA tyrosine phosphorylation. Of the 16 negative CagA tyrosine phosphorylation isolates, intact tyrosine phosphorylation motifs were found in 15 isolates. Conclusions. CagA tyrosine phosphorylation, which is significantly greater in strains from gastric adenocarcinoma patients, may play a role in gastric carcinogenesis, and could be a better marker of more virulent strains than the cag pathogenicity island in Asia, where the cag pathogenicity island is present in nearly all H. pylori strains. Sequence diversity of tyrosine phosphorylation motifs on CagA was not related to the presence of tyrosine phosphorylation. The absence of tyrosine phosphorylation motif might result in negative tyrosine phosphorylation phenotypes, but such motifs are not the sole factors associated with CagA tyrosine phosphorylation.     

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.     

Environmental post-processing increases the adhesion strength of mussel byssus adhesive

Marine mussels (Mytilus trossulus) attach to a wide variety of surfaces underwater using a protein adhesive that is cured by the surrounding seawater environment. In this study, the influence of environmental post-processing on adhesion strength was investigated by aging adhesive plaques in a range of seawater pH conditions. Plaques took 8–12 days to achieve full strength at pH 8, nearly doubling in adhesion strength (+94%) and increasing the work required to dislodge (+59%). Holding plaques in low pH conditions prevented strengthening, causing the material to tear more frequently under tension. The timescale of strengthening is consistent with the conversion of DOPA to DOPA-quinone, a pH dependent process that promotes cross-linking between adhesive proteins. The precise arrangement of DOPA containing proteins away from the adhesive-substratum interface emphasizes the role that structural organization can have on function, an insight that could lead to the design of better synthetic adhesives and metal-coordinating hydrogels.     

Anti-adhesive properties of fish tropomyosins

Aims:  We have recently found that preconditioning of stainless steel surfaces with an aqueous fish muscle extract can significantly impede bacterial adhesion. The purpose of this study was to identify and characterize the primary components associated with this bacteria-repelling effect.
Methods and Results:  The anti-adhesive activity was assayed against Escherchia coli K-12, and bacterial adhesion was quantified by crystal violet staining and sonication methods. Proteolytic digestion, elution and fractionation experiments revealed that the anti-adhesive activity of the extract was linked to the formation of a proteinaceous conditioning film composed primarily of fish tropomyosins. These fibrous proteins formed a considerable anti-adhesive conditioning layer on and reduced bacterial adhesion to several different materials including polystyrene, vinyl plastic, stainless steel and glass. The protein adsorption profiles obtained from the various materials did not differ significantly, but elution was often incomplete making minor qualitative/quantitative differences indiscernible.
Conclusions:  The data highlights the significance of protein conditioning films on bacterial adhesion and emphasizes the importance of substratum's physiochemical properties and exposure time with regards to protein adsorption/elution efficiency and subsequent bacterial adhesion.
Significance and Impact of the Study:  Fish tropomyosin-coatings could potentially offer a nontoxic and relatively inexpensive measure of reducing bacterial colonization of inert surfaces.