Surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity

The synthesis of extracellular molecules such as biosurfactants should have major consequences on bacterial adhesion. These molecules may be adsorbed on surfaces and modify their hydrophobicities. Certain strains of Bacillus subtilis synthesize the lipopeptides, which exhibit antibiotic and surface active properties. In this study the high-performance liquid chromatography (HPLC) analysis of the culture supernatants of the seven B. subtilis strains, showed that the lipopeptide profile varied greatly according to the strain. Among the three lipopeptide types, only iturin A was produced by all B. subtilis strains. Bacterial hydrophobicity, evaluated by the water contact angle measurements and the hydrophobic interaction chromatography, varied according to the strain. Two strains (ATCC 15476 and ATCC 15811) showing extreme behaviors in term of hydrophobicity were selected to study surfactin and iturin A effects on bacterial hydrophobicity. The two lipopeptides modified the B. subtilis surface hydrophobicity. Their effects varied according to the bacterial surface hydrophobic character, the lipopeptide type and the concentration. Lipopeptide adsorption increased the hydrophobicity of the hydrophilic strain but decreased that of the hydrophobic. Comparison of lipopeptide effects on B. subtilis surface hydrophobicity showed that surfactin was more effective than iturin A for the two strains tested.     

Role of hydrophobicity in bacterial adherence to carbon nanostructures and biofilm formation

The role of cell and surface hydrophobicity in the adherence of the waterborne bacterium Mycobacterium smegmatis to nanostructures and biofilm formation was investigated. Carbon nanostructures (CNs) were synthesized using a flame reactor and deposited on stainless steel grids and foils, and on silicon wafers that had different initial surface hydrophobicities. Surface hydrophobicity was measured as the contact angle of water droplets. The surfaces were incubated in suspensions of isogenic hydrophobic and hydrophilic strains of M. smegmatis and temporal measurements of the numbers of adherent cells were made. The hydrophobic, rough mutant of M. smegmatis adhered more readily and formed denser biofilms on all surfaces compared to its hydrophilic, smooth parent. Biofilm formation led to alterations in the hydrophobicity of the substratum surfaces, demonstrating that bacterial cells attached to CNs are capable of modifying the surface characteristics.     

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.     

Characterisation of Curli Production, Cell Surface Hydrophobicity, Autoaggregation and Attachment Behaviour of Escherichia coli O157

Escherichia coli O157 are an important group of foodborne pathogens with the ability to attach to materials commonly used in food processing environments such as slightly hydrophilic stainless steel. The aim of this study was to characterise six E. coli isolates, including five E. coli O157, for curli production, autoaggregation, hydrophobicity and attachment to highly hydrophilic glass and hydrophobic Teflon^®. Curli production and autoaggregation were determined using absorbance assays; hydrophobicity by bacterial adherence to hydrocarbons, hydrophobic interaction chromatography and contact angle measurements; and attachment using epifluorescence microscopy. Curli production varied between strains and for some strains correlated with autoaggregation. Curli production correlated with decreased hydrophobicity for two strains. Four of the six isolates increased attachment to glass, but decreased attachment to Teflon^® with increased curli production. In contrast, one of the six isolates decreased attachment to glass, but increased attachment to Teflon^® with increasing curli production. Curli production by the remaining isolate did not correlate with hydrophobicity or attachment. Attachment of some E. coli, including E. coli O157, to abiotic surfaces may be influenced by curli production, autoaggregation and hydrophobicity. However, for other strains, a variety of factors may be of greater influence on these properties and ability to attach to abiotic surfaces. This study highlights the complexity of bacterial surface properties and their relationship with bacterial attachment.     

Study of microbial adhesion on some wood species: Theoretical prediction

The initial interaction between microorganisms and substrata is mediated by physicochemical forces, which in turn originate from the physicochemical surface properties of both interacting phases. In this context, we have determined the physicochemical proprieties of all microorganisms isolated from cedar wood decay in an old monument at the Medina of Fez-Morocco. The cedar wood was also assayed in terms of hydrophobicity and electron donor-electron acceptor (acid-base) properties. Investigations of these two aspects were performed by contact angles measurements via sessile drop technique. Except Bacillus subtilis strain (Giwi < 0), all strains studied showed positive values of the degree of hydrophobicity (Giwi > 0) and can therefore be considered as hydrophilic while cedar wood revealed a hydrophobic character (Giwi = 58.81 mJ m⁻²). All microbial strains were predominantly electron donor. The results show also that all strains were weak electron acceptors. Cedar wood exhibits a weak electron donor/acceptor character. Based on the thermodynamic approach, the Lifshitz-van der Waals interaction free energy, the acid-basic interactions free energy, the total interaction free energy between the microbial cells and six different wood species (cedar, oak, beech, ash, pine and teak) in aqueous media was calculated and used to predict which microbial strains have a higher ability to adhere to wooden surfaces. Except of weak wood, for all the situations studied, generalizations concerning the adhesion of the microbiata on wood species cannot be made and the microbial adhesion on wooden substrata was dependent on wood species and microorganisms tested.     

Hydrophobicity and surface electrostatic charge of conidia of the mycoparasitic <Emphasis Type="Italic">Trichoderma</Emphasis> species

The present investigation was done to understand the fungal-fungal interactions mechanisms based on level of nonspecific adhesion of a potential fungal mycoparasite (Trichoderma) to their fungal host (Macrophomina phaseolina). The relative cell surface hydrophobicity (CSH) and cell surface electrostatic charge (CSEC) of 29 isolates of Trichoderma species, analyzed by bacterial adhesion to hydrocarbon (BATH), hydrophobic interaction chromatography (HIC), microelectrophoresis and contact angle, revealed a large degree of variability. CSH and CSEC of conidia depended on culture age, pH and temperature. Maximum CSH and CSEC were recorded in 25–28 °C range, and both declined significantly with increasing temperature. Isolate Trichoderma hazianum (Th)-23/98 expressed surface hydrophobicity at 25–28 °C and hydrophilicity at 40 °C. Surface hydrophobicity of the isolate was susceptible to various proteases (trypsin, pepsin, proteinase k and a-chymotrypsin) and inhibitors (SDS, mercaptoethanol and Triton X-100) and a significant reduction in CSH was recorded in hydrophobic conidia. Hydrophilic conidia remained more or less unaffected by such treatments. SDS-PAGE analysis of the hydrophobic and hydrophilic conidia exhibited several protein bands in the 25 to 61 kDa range. However, each protein population contained one protein that was not observed in the other population. For hydrophobic conidia, the unique protein had an apparent molecular mass of 49 kDa, while the unique protein associated with hydrophilic conidia had a molecular mass of 61 kDa. Our findings suggest that CSH and CSEC of mycoparasitic Trichoderma may contribute to non-specific adhesion on to the sclerotial surfaces of Macrophomina phaseolina that may be influenced by growth and environmental conditions.     

Effects of pantolactone and butyrolaectone on the pleiotropic phenotypes of lon mutants and on thermal induction of the SOS phenomena in a tif mutant of Escherichia coli K12

Hydrophobic and charge-charge interactions of Salmonella typhimirium and Serratia marcescens were determined and related to their content of fimbriae and lipopolysaccharide (LPS). The cell surface structures were characterized with hydrophobic interaction chromatography (HIC), electrostatic interaction chromatography (ESIC) and particle electrophoresis measurements. The degree of interaction at the air-water interface was tested using a monolayered lipid film applied to an aqueous surface. The cell surface hydrophobicity of S. typhimurium in the presence of fimbriae was less in smooth than in rought bacteria. Examination of a series of rough mutants of S. typhimurium indicates that reduction of the O-side chain and core oligosaccharides was correlated with increased cell hydrophobicity. The enrichment factors at the air-water interface were significantly higher for fimbriated than for non-fimbriated S. typhimurium cells. Fimbriated S. marcescens cells were less hydrophobic and adhered to a lesser degree at the air-water surface than non-fimbriated counterparts. Electrophoretic measurements and adsorption to ion exchangers gives different information about the surface charge of bacteria. The latter technique gives the interaction between localized charged surfaces.Abbreviations HIC hydrophobic interaction chromatography - ESIC electrostatic interaction chromatography - LPS lipopolysaccharide - PBS phosphate buffered saline solution     

Effect of curli expression and hydrophobicity of Escherichia coli O157:H7 on attachment to fresh produce surfaces

Aim: To investigate the effect of curli expression on cell hydrophobicity, biofilm formation and attachment to cut and intact fresh produce surfaces. Methods and Results: Five Escherichia coli O157:H7 strains were evaluated for curli expression, hydrophobicity, biofilm formation and attachment to intact and cut fresh produce (cabbage, iceberg lettuce and Romaine lettuce) leaves. Biofilm formation was stronger when E. coli O157:H7 were grown in diluted tryptic soy broth (1 : 10). In general, strong curli‐expressing E. coli O157:H7 strains 4406 and 4407 were more hydrophobic and attached to cabbage and iceberg lettuce surfaces at significantly higher numbers than other weak curli‐expressing strains. Overall, E. coli O157:H7 populations attached to cabbage and lettuce (iceberg and Romaine) surfaces were similar (P > 0·05), indicating produce surfaces did not affect (P < 0·05) bacterial attachment. All E. coli O157:H7 strains attached rapidly on intact and cut produce surfaces. Escherichia coli O157:H7 attached preferentially to cut surfaces of all produce types; however, the difference between E. coli O157:H7 populations attached to intact and cut surfaces was not significant (P > 0·05) in most cases. Escherichia coli O157:H7 attachment and attachment strength (S_R) to intact and cut produce surfaces increased with time. Conclusions: Curli‐producing E. coli O157:H7 strains attach at higher numbers to produce surfaces. Increased attachment of E. coli O157:H7 on cut surfaces emphasizes the need for an effective produce wash to kill E. coli O157:H7 on produce. Significance and Impact of the Study: Understanding the attachment mechanisms of E. coli O157:H7 to produce surfaces will aid in developing new intervention strategies to prevent produce outbreaks.     

Acceleration of fungal spore production by embedding a hydrophobic polymer net in a nutrient agar plate

A simple and novel procedure for the acceleration of fungal spore production was developed. A net of hydrophobic polymer such as polypropylene (PP) and polytetrafluoroethylene (PTFE) was embedded in a nutrient agar plate, and effect of the polymer net on spore production by 6 fungal strains, such as Aspergillus terreus, Penicillium multicolor, and Trichoderma virens were estimated. The effect of hydrophobic polymer net was insufficient in a liquid-surface immobilization (LSI) system with fungal cells immobilized on a ballooned microsphere layer formed on a liquid medium surface. On the other hand, the embedding of a PTFE net in an agar plate remarkably enhanced the spore production in all 6 strains tested to produce 2.0–8.5 × 10⁷ spores/cm²-agar plate surface. Especially, the spore production by A. terreus ATCC 20542 in the presence of a PTFE net was 7.7 times as much than that in no net. Positive correlations between the hydrophobicity of net and the spore production were observed in all 6 strains (R², 0.653–0.999).     

Cell surface hydrophobicity ofStaphylococcus aureus measured by the salt aggregation test (SAT)

Well-defined laboratory strains as well as 72 clinical strains ofStaphylococcus aureus isolated from bovine mastitis were investigated for surface hydrophobicity by the salt aggregation test (SAT).Staphylococcus aureus strain Cowan 1, rich in protein A and fibronectin-binding surface proteins, was found to show high surface hydrophobicity, whereas strain Wood 46, deficient in these surface proteins, showed low surface hydrophobicity. SAT showed a significant difference in surface hydrophobicity (P<0.001) between protein A-positive and A-negative strains measured by ²-test analysis. Comparison of SAT values with results obtained from hydrophobic interaction chromatography (HIC) showed a good correlation (P<0.025). A high-level protein-A-producing mutant (SA 113prA-3) showed increased surface hydrophobicity as compared with the parent strain (SA 113), whereas ten protein-A-negative mutants showed low surface hydrophobicity in SAT. Of the 72 clinical isolates tested by SAT, 47 (65%) showed autoaggregating properties, i.e., the strains aggregated even in isotonic buffers. Tween 80 (1% vol/vol) and ethylene glycol (50% vol/vol) prevented autoaggregation of some hydrophobic strains aggregating in phosphate-buffered saline. However, 2M of a chaotropic agent (NaSCN) was more efficient in preventing autoaggregation of the strains tested. Heating of cell suspensions to 80°C or 100°C as well as trypsin andStreptomyces griseus protease treatment generally caused a decrease in the cell surface hydrophobicity. This indicates that protein A, fibronectin-binding proteins, and probably other as yet unidentified proteins contribute to the high surface hydrophobicity of most strains isolated from bovine mastitis.     

Surface Hydrophobicity of Culture and Water Biofilm of <Emphasis Type="Italic">Penicillium</Emphasis> spp.

Fungal surface hydrophobicity is involved in several functions in fungal growth and development. Water contact angles measurement has been used as a direct and simple approach for its characterisation in solid cultures. Microsphere adhesion assay is said to be the best method to assess cell hydrophobicity of filamentous fungi. This study aimed to apply these two methods to study hydrophobicity of Penicillium expansum and Penicillium brevicompactum grown as mycelial mats in solid culture, liquid culture and water biofilms. As result, both species in solid cultures were classified as hydrophobic with contact angles ≥90o, but in liquid cultures and water biofilms showed different levels of hydrophobicity when microsphere adhesion assay was applied. In addition, was found that biofilms have specific hydrophobic hyphae which may be involved in fungal ecological functions.     

Cell hydrophobicity of <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. and <Emphasis Type="Italic">Bacillus</Emphasis> spp. bacteria and hydrocarbon biodegradation in the presence of Quillaya saponin

Biodegradation and hydrophobicity of Pseudomonas spp. and Bacillus spp. strains were tested at different concentrations of the biosurfactant Quillaya saponin. A model mixture of hydrocarbon (dodecane and hexadecane) was used for estimating the influence of surfactants on biodegradation. The bacterial adhesion to hydrocarbon method for determination of bacterial cell surface hydrophobicity was exploited. Among the tested bacterial strains the higher hydrophobicity was noticed for Pseudomonas aeruginosa TK. The hydrophobicity of this strain was 84%. The highest hydrocarbon biodegradation was observed for P. aeruginosa TK (49%) and Bacillus subtilis (35%) strains after 7 days of experiments. Generally the addition of Quillaya saponin increased hydrocarbon biodegradation remarkably. The optimal concentration proved to be 80 mg l⁻¹. The degree of hydrocarbon biodegradation was 75% for P. aeruginosa TK after the addition of saponin. However the most significant increase in biodegradation after addition of Quillaya saponin was in the case of P. aeruginosa 25 and Pseudomonas putida (the increase of biodegradation from 21 to 52% and from 31 to 66%, respectively). It is worth mentioning that decrease of hydrophobicity is correlated with the best biodegradation by P. aeruginosa strain. For the remaining strains, no significant hydrophobicity changes in relation to the system without surfactant were noticed.     

Effect of different Bacillus subtilis lipopeptides on surface hydrophobicity and adhesion of Bacillus cereus 98/4 spores to stainless steel and Teflon

Various lipopeptides produced by Bacillus subtilis were examined for their ability to modify the surface hydrophobicity of two substrata, stainless steel (SS) and Teflon. These modifications were evaluated by water contact angle measurements. The effects depended on the lipopeptide, its concentration, and the tested substratum. Treatment of SS with different concentrations of surfactin S1 showed an increase of the hydrophobicity between 1 and 100 mg l^?1. On the same substratum, fengycin increased hydrophobicity up to its critical micelle concentration (6.25 mg l^?1). With higher concentrations of fengycin, hydrophobicity decreased. Surfactin, mycosubtilin, and iturin A decreased hydrophobicity on Teflon. The different effects of these three families of lipopeptides were related to their structural differences. A good correlation was shown between hydrophobicity modifications of surfaces and the attachment of B. cereus 98/4 spores. Enhancement in the hydrophobicity of the surfaces increased the number of adhering spores.     

Role of Cell Hydrophobicity on Colony Formation in Microcystis (Cyanobacteria)

Colony formation is highly import ant for the competitive advantage of the cyanobacterium Microcystis over other phytoplankton species. The laboratory‐grown colonial Microcystis strains isolated from Lake Taihu (China) maintained colonial forms under the low light condition (10 μE m^–2 s^–1). The cell surface hydrophobicities of the Microcystis colonies were measured by cyanobacterial adherence to xylene in comparison with unicellular Microcystis strains. The cells of the tested colonial strains were all hydrophobic, while the cells of the tested unicellular strains were all hydrophilic. Incubation under the higher light condition (75 μE m^–2 s^–1) leaded to the significant decrease in the cell hydrophobicities of the colonial Microcystis and the transition from colonial forms to unicellular forms. These findings indicated that the cell hydrophobicity of Microcystis may play a role in cell‐cell adherence and colony formation. Phosphate‐limitation, nitrate‐limitation and pH did not affect cell hydrophobicities of colonial Microcystis. Treatment with proteolytic enzymes had no effect on the cell hydrophobicity, indicating that cell surface proteins did not contribute to high cell hydrophobicity. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Hydrophobic adsorptive and hemagglutinating properties ofEscherichia coli possessing colonization factor antigens (CFA/I or CFA/II), type 1 pili,or other pili

Hydrophobic and hemagglutinating activities of piliated enterotoxigenicEscherichia coli possessing colonization factor antigens (CFA)/I and putative CFA/II, strains with type 1 pili, and piliated strains of nonenterotoxigenicE. coli from urinary tract infections were compared. When passed through columns of hydrophobic Phenyl Sepharose in the presence of buffered ammonium sulfate, the strains with CFA adsorbed most strongly. Similarly, the CFA strains showed a tendency to autoagglutinate at a lower (NH₄)₂SO₄ concentration than the other strains studied. The degree of hydrophobicity of the strains tested is in the order CFA/I>CFA/II>type 1 pili>urinary tract strains. Rough variants ofE. coli strains were more hydrophobic than their smooth parents. Electron microscopy showed large numbers of pili on CFA strains, whereas type 1 piliated strains possessed fewer pili. CFA-negative clones possessed few or no, pili and did not adsorb to the gel. A highly piliated mutant strain (PAK/2PfS) ofPseudomonas aeruginosa bound to the Phenyl Sepharose while the poorly piliated wild-type strains did not. Strains, lost their adsorptive capacity after blending, sonication, heating, or trypsin treatment. It is concluded that the hydrophobicity of enteric organisms, as measured by hydrophobic interaction chromatography, is a function of the type and number of pili on the cell surface.     

Significance of the rdar and bdar morphotypes in the hydrophobicity and attachment to abiotic surfaces of Salmonella Sofia and other poultry-associated Salmonella serovars

Aims: To investigate the relative role of the red dry and rough (rdar) and brown dry and rough (bdar) morphotypes on hydrophobicity and ability to attach to abiotic surfaces of poultry‐associated Salmonella strains with a focus on S. Sofia. Methods and Results: Cellulose synthase gene null mutants were constructed in five Salmonella strains converting them from rdar to bdar morphotypes. One S. Sofia null mutant displayed reduced hydrophobicity and attachment to Teflon^® relative to its parent strain. The S. Virchow and S. Infantis null mutants attached less well to glass relative to their parent strains. Conclusions: The rdar or bdar morphotype may influence S. Sofia persistence but did not explain why bdar strains predominate in this serotype. Significance and Impact of the Study: This work provides some insight into why some Salmonella strains survive in poultry environments and may ultimately contribute to their control.     

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.     

The hydrophobicity of bacteria — An important factor in their initial adhesion at the air-water inteface

Bacteria isolated from the surface and the subsurface water at four stations along the Swedish west coast were assessed for their hydrophobicity with hydrophobic interaction chromatography (HIC). The surface bacteria were sampled by the Teflon sheet technique. [³H]-l-leucine metabolically labeled isolates were run on a column packed with Octyl-Sepharose CL-4B gel. The relative hydrophobicity of the bacteria was expressed as the ratio, g/e, between the radioactivity of the gel and the eluate. The results revealed a positive correlation between the degree of enrichment of bacteria at the surface and their hydrophobicity. The subsurface bacteria exhibited a broader spectrum of g/e-values than the surface bacteria. The initial adhesion of bacteria to the surface microlayer depends on several factors of which the hydrophobic interaction may be one of the most important.Abbreviations HIC hydrophobic interaction chromatography - NSS nine salt solution     

Selective isolation of bacteria from soil with hydrophobic materials

Bacterial strains having a hydrophobic cell surface have often been considered as degraders of hydrophobic organic pollutants in soil. In this study, bacterial strains were isolated using hydrophobic materials from 12 soil samples, and their cell surface hydrophobicity was determined by evaluating their adherence to n-hexane. Bacterial strains isolated using polytetrafluoroethylene (PTFE) membrane were more hydrophobic on an average than those isolated with styrene–divinylbenzene (DVB) particles or octadecylsilyl silica gel (ODS) particles. Strains closely related to Burkholderia cepacia could be selectively isolated using the PTFE membrane; those closely related to Ralstonia pickettii, using ODS and DVB particles; and those closely related to B. fungorum, using DVB. These results indicate that bacterial strains having a hydrophobic cell surface or within certain phyla can be selectively isolated from soils using hydrophobic materials, and that this isolation method would be useful for collecting candidates for bioremediation of hydrophobic pollutants.     

Application of three-dimensional molecular hydrophobicity potential to the analysis of spatial organization of membrane domains in proteins: I. Hydrophobic properties of transmembrane segments of Na+, K+-ATPase

A new computer-aided molecular modeling approach based on the concept of three-dimensional (3D) molecular hydrophobicity potential has been developed to calculate the spatial organization of intramembrane domains in proteins. The method has been tested by calculating the arrangement of membrane-spanning segments in the photoreaction center ofRhodopseudomonas viridis and comparing the results obtained with those derived from the X-ray data. We have applied this computational procedure to the analysis of interhelical packing in membrane moiety of Na⁺, K⁺-ATPase. The work consists of three parts. In Part I, 3D distributions of electrostatic and molecular hydrophobicity potentials on the surfaces of transmembrane helical peptides were computed and visualized. The hydrophobic and electrostatic properties of helices are discussed from the point of view of their possible arrangement within the protein molecule. Interlocation of helical segments connected with short extramembrane loops found by means of optimization of their hydrophobic/hydrophilic contacts is considered in Part II. The most probable 3D model of packing of helical peptides in the membrane domain of Na⁺, K⁺-ATPase is discussed in the final part of the work.