Microbial Cell Surface Research Articles

Resistance to high temperatures salmonellosis circulating in Kemerovo region

Anti-lactoferrin activity (AlfA) is one of the factors contributing to the persistence of V. cholerae in human body in formation of human vibriocarriers. It is shown that in the AlfA mechanism cholera vibrio lectins are participating as an initial link specifically binding to lactoferrin at the surface of microbial cells, providing for subsequent cleavage of this protein with the help of V. cholerae exoproteases. Lectins are proteins or glycoproteins of non-immune class, capable to reversibly bind to the carbohydrate fraction of glycoconjugates without breaking the covalent structure of any of the recognizable glycosyl ligands. As glycoproteins, lectins ensure the complementary highly specific carbohydrate-protein binding of the active sites of target cells (namely lactoferrin). It is proved that all V. cholerae El Tor and Bengal strains possess hexose-specific lectin receptors (especially mannose). Atoxigenic V. cholerae O1 and O139 strains possess, in addition to hexose receptors, lectins specific to aminosugars.

Competitive adsorption in a ternary system of toxic metals and rare earth elements using Desmodesmus multivariabilis: empirical and kinetic modelling

The presence of competing metals in industrial wastewater creates problems of selectivity for binding sites during biosorption of cationic metallic species on microbial cell surfaces. For this reason, modelling of adsorption and uptake of multimetal solutions based on observation of performance of single metal species since this neglects the competition for reactive sites typical in multimetal solutions. In this study, a culture of the green alga Desmodesmus multivariabilis previously isolated from eutrophic freshwater was used as a biosorbent for adsorption/desorption of different metals in a mixed metal solution under varying environmental conditions. The Extended Langmuir Model (ELM) and Combined Langmuir and Freundlich Model (CLFM) were used to evaluate the data from a ternary metallic system. AQUASIM was used for optimization and simulation of kinetic data in ternary systems. The ELM performed better than CLFM with correlation coefficient of approximately 0.99. The highest uptake was observed for thallium with q max = 909.09 mg g−1 in single metal solution. This value was reduced to q max = 77.278 mg g−1 in the presence of other metals which demonstrated the competitive effects of other metal ions in solution. The q max value of lanthanum (La) decreased from 100 in single metals to 71.3 mg g−1 in multimetal solution; whereas, the q max = of cadmium (Cd) decreased from 48.5 to 40.1 mg g−1. Recovery of metals by D. multivariabilis was the highest for La at 90.92% followed by Cd at 90.15%. Competitive inhibition models provided a better insight on the potential for treatment of actual industrial wastewater with varying environmental factors.

Sorption Behavior of Np(V) on Microbe Pure Culture and Consortia

The sorption of Np(V) by microbe consortia and by a single pure culture of the Fe-reducing bacterium Shewanella putrefaciens was studied at pH levels between 3 and 7 under resting cell conditions. The sorption of Np(V) by S. putrefaciens obtained under inert conditions and by the consortia in the aerated condition was higher than that by S. putrefaciens under aerobic conditions at low pH below 5, strongly suggesting the presence of another mechanism other than the adsorption of Np(V) on the microbial cell surface, i.e., by reduction to Np(IV).

Molecular cloning and characterization of antimicrobial peptides from skin of Hylarana guentheri.

The cDNAs encoding antimicrobial peptides (AMPs) in the skin of Hylarana guentheri were identified, namely temporin (five peptides, termed temporin-GHa-GHd and temporin-GUa), brevinin-1 (one peptide, brevinin-1GUb), and brevinin-2 (eight peptides, brevinin-2GHd-2GHj, and brevinin-2GHb). Eleven of the 14 peptides have novel primary structures. Synthesized temporin GHa-GHd have broad-spectrum antimicrobial activities against Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli, Vibrio alginolyticus, and Pseudomonas aeruginosa), as well as fungus (Candida albicans). Among these tested strains, S. aureus was the most sensitive to temporin-GHa-GHd with minimum inhibitory concentration (MIC) values between 6.8 and 12.9 μM. They also exhibited antimicrobial activities against Methicillin-resistant S. aureus with the MIC ranging from 12.7 to 51.7 μM. Interestingly, secondary structure prediction shows that there is no α-helix in temporin-GHb, which illustrates that α-helix is not required for the antimicrobial activity of temporin-GHb. NaCl (at final concentrations of 0.15-2 M) decreased the antimicrobial activity of temporin-GHa-GHd slightly, while human serum and S. aureus V8 proteinase had no effect on the antimicrobial activity. Scanning electron microscopy images of E. coli and S. aureus showed that the surface of microbial cells was considerably rough and shrived after 1 h of treatment with temporin-GHa-GHd at 37°C. The stabilities of temporin-GHa-GHd in human serum or in S. aureus V8 proteinase make them to be promising candidates of novel antimicrobial agents or models for the development of novel AMPs.

Influence of silver nanoparticles and liberated silver ions on nitrifying sludge: ammonia oxidation inhibitory kinetics and mechanism.

Silver nanoparticles (AgNPs) are widely used in commercial products because of their excellent antimicrobial activity. Entrance of AgNPs and its released Ag ions (Ag+) into wastewater treatment plants could harm ammonia oxidation (AO) process resulting in environmental problems. This study investigated inhibitory kinetics and mechanism of AO from nitrifying sludge influenced by AgNPs and Ag+. The findings demonstrated that AgNPs and Ag+ adversely influenced on AO. Silver ions were more toxic to AO than AgNPs, which was indicated by the lower inhibitory constant (K i ) of 0.29mg/L compared to that of AgNPs (K i of 73.5mg/L). Over the experimental period of 60h, AgNPs at 1, 10, and 100mg/L released Ag+ in the average concentrations of 0.059, 0.171, and 0.503mg/L, respectively. Silver nanoparticles of 1-100mg/L inhibited AO by 45-74%, whereas Ag+ of 0.05-0.50mg/L inhibited AO by 53-94%. This suggested that the AgNP toxicity mainly derived from the liberated Ag+. Scanning electron microscopy results revealed that AgNPs attached on microbial cell surfaces, and both AgNPs and Ag+ induced cell morphological change from rod shape to shorter rod shape. Transmission electron microscopy showed that AgNPs and Ag+ diminished the thickness of the outer layer and reduced the density of internal parts of the exposed microbial cells, which could be the reasons for the morphology change. Live/dead results also confirmed that AgNPs and Ag+ damaged membrane integrity of cells in the nitrifying sludge. This study suggested that the primary mechanism for toxicity of AgNPs was the liberation of Ag+ and then both of silver species caused cell death.

Chlorination-mediated EPS excretion shapes early-stage biofilm formation in drinking water systems

Abstract Microbial surface adhesion to surfaces and subsequent biofilm establishment are ubiquitous in drinking water systems, which often contribute to deteriorated water quality. Disinfectants are common agents applied to drinking water controlling microbial propagation, yet the underlying mechanisms of how disinfectants function to regulate microbial activity and thereby biofilm development remains elusive. We experimentally studied the effects of chlorination on extracellular polymeric substance (EPS) production, and its impacts on early-stage biofilm formation in a model drinking water system. Results showed that low-level chlorine (≤ 1.0 mg/L) stimulated microbial EPS (especially of proteins) excretion that favored early-stage biofilm formation. Microbes experiencing higher chlorination (>1.0 mg/L) exhibited clearly suppressed growth associated with reduced EPS release, consequently yielding less biofilm formation. Removal of cell-attached proteins and polysaccharides diminished biofilm formation, which highlighted the critical role of EPS (especially protein components) in biofilm development. A negative correlation between chlorination-mediated microbial protein production and cell surface charge suggested that chlorine disinfection may modify cell surface properties through regulation of microbial EPS excretion and thereby mediate biofilm formation. With these quantitative estimations, this study provides novel insights into how chlorination-mediated EPS excretion shapes early-stage biofilm formation, which is essential for practical functioning of drinking water systems.

Recombinant Human REG I<i>α</i> Aggregates <i>Staphylococcus aureus</i>—Exhibits a Lectin-Like Function

Staphylococcus aureus is pathogenic to humans with worldwide health care concern due to its ability to evade the immune system and develop resistance to multiple drugs. Reg family proteins are C-type lectins with antimicrobial properties. Bacterial aggregation through binding to microbial cell surface sugar and/or lipid moieties is key mechanism employed in the process. In the present study we have analysed the antimicrobial effect of human REG Iα on S. aureus. Aggregation of mid-log phase culture of S. aureus was observed in presence of purified recombinant REG Iα. Therefore REG Iα can be applied in eliminating S. aureus infections in humans.

High-resolution imaging of the microbial cell surface.

Microorganisms, or microbes, can function as threatening pathogens that cause disease in humans, animals, and plants; however, they also act as litter decomposers in natural ecosystems. As the outermost barrier and interface with the environment, the microbial cell surface is crucial for cell-to-cell communication and is a potential target of chemotherapeutic agents. Surface ultrastructures of microbial cells have typically been observed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Owing to its characteristics of low-temperature specimen preparation and superb resolution (down to 1 nm), cryo-field emission SEM has revealed paired rodlets, referred to as hydrophobins, on the cell walls of bacteria and fungi. Recent technological advances in AFM have enabled high-speed live cell imaging in liquid at the nanoscale level, leading to clear visualization of cell-drug interactions. Platinum-carbon replicas from freeze-fractured fungal spores have been observed using transmission electron microscopy, revealing hydrophobins with varying dimensions. In addition, AFM has been used to resolve bacteriophages in their free state and during infection of bacterial cells. Various microscopy techniques with enhanced spatial resolution, imaging speed, and versatile specimen preparation are being used to document cellular structures and events, thus addressing unanswered biological questions.

Surface display on lactic acid bacteria without genetic modification: strategies and applications.

Microbial cell surface display has attracted greater attention than ever and has numerous potential applications in biotechnology. With the safety and probiotic properties, lactic acid bacteria (LAB) have been used widely in food and industrial applications. In order to circumvent using genetically modified microorganisms which face low public acceptance and severe regulatory scrutiny, surface-engineered LAB without genetical modification are more preferred. According to the way used to obtain the fusion protein containing the passenger molecule and anchoring domain, the genetic or chemical approaches can be used to construct these surface-engineered LAB. In addition to the viable wide-type LAB, non-living bacterial-like particles (BLP) can be attached by these fusion proteins added from outside. Compared to the living LAB, BLP have a higher binding capacity and less anticarrier response. Mucosal vaccines are the predominant application of these surface-engineered LAB with no genetical modification.

Reversible off and on switching of prion infectivity via removing and reinstalling prion sialylation.

The innate immune system provides the first line of defense against pathogens. To recognize pathogens, this system detects a number of molecular features that discriminate pathogens from host cells, including terminal sialylation of cell surface glycans. Mammalian cell surfaces, but generally not microbial cell surfaces, have sialylated glycans. Prions or PrPSc are proteinaceous pathogens that lack coding nucleic acids but do possess sialylated glycans. We proposed that sialylation of PrPSc is essential for evading innate immunity and infecting a host. In this study, the sialylation status of PrPSc was reduced by replicating PrPSc in serial Protein Misfolding Cyclic Amplification using sialidase-treated PrPC substrate and then restored to original levels by replication using non-treated substrate. Upon intracerebral administration, all animals that received PrPSc with original or restored sialylation levels were infected, whereas none of the animals that received PrPSc with reduced sialylation were infected. Moreover, brains and spleens of animals from the latter group were completely cleared of prions. The current work established that the ability of prions to infect the host via intracerebral administration depends on PrPSc sialylation status. Remarkably, PrPSc infectivity could be switched off and on in a reversible manner by first removing and then restoring PrPSc sialylation.

Involvement of a versatile pattern recognition receptor, apolipophorin-III in prophenoloxidase activation and antibacterial defense of the Chinese oak silkworm, Antheraea pernyi

Apolipophorin-III (apoLp-III) is an exchangeable apolipoprotein found in many insect species and functions as a lipid transport vehicle. Recent studies have shown that apoLp-III is a multifunctional molecule involved in not only lipid transportation but also innate immune responses. In the present study, the pattern recognition properties of Antheraea pernyi apoLp-III were investigated. Recombinant Ap-apoLp-III was bound to different species of microbes and further study showed the rAp-apoLp-III is capable of interacting with pathogen associated molecular patterns (PAMPs) on the microbial cell surface. In addition, an Ap-apoLp-III/PAMP mixture stimulated the prophenoloxidase (PPO) activation of A. pernyi hemolymph in vitro, to a greater extent than PAMP alone while Ap-apoLp-III itself failed to activate the PPO system, indicating that Ap-apoLp-III up-regulates PPO activation by combining with PAMP. After pathogen invasion following an injection of Staphylococcus aureus, RNAi-mediated silencing of apoLp-III decreased the transcriptional abundance of three antimicrobial peptide genes. These data suggest that apoLp-III is a versatile pattern recognition receptor and may play important roles in the innate immune responses of Antheraea pernyi.

Current Technological Improvements in Enzymes toward Their Biotechnological Applications.

Enzymes from extremophiles are creating interest among researchers due to their unique properties and the enormous power of catalysis at extreme conditions. Since community demands are getting more intensified, therefore, researchers are applying various approaches viz. metagenomics to increase the database of extremophilic species. Furthermore, the innovations are being made in the naturally occurring enzymes utilizing various tools of recombinant DNA technology and protein engineering, which allows redesigning of the enzymes for its better fitment into the process. In this review, we discuss the biochemical constraints of psychrophiles during survival at the lower temperature. We summarize the current knowledge about the sources of such enzymes and their in vitro modification through mutagenesis to explore their biotechnological potential. Finally, we recap the microbial cell surface display to enhance the efficiency of the process in cost effective way.

Proof of concept for the simplified breakdown of cellulose by combining Pseudomonas putida strains with surface displayed thermophilic endocellulase, exocellulase and β-glucosidase.

BackgroundThe production and employment of cellulases still represents an economic bottleneck in the conversion of lignocellulosic biomass to biofuels and other biocommodities. This process could be simplified by displaying the necessary enzymes on a microbial cell surface. Such an approach, however, requires an appropriate host organism which on the one hand can withstand the rough environment coming along with lignocellulose hydrolysis, and on the other hand does not consume the generated glucose so that it remains available for subsequent fermentation steps.ResultsThe robust soil bacterium Pseudomonas putida showed a strongly reduced uptake of glucose above a temperature of 50 °C, while remaining structurally intact hence recyclable, which makes it suitable for cellulose hydrolysis at elevated temperatures. Consequently, three complementary, thermophilic cellulases from Ruminiclostridium thermocellum were displayed on the surface of the bacterium. All three enzymes retained their activity on the cell surface. A mixture of three strains displaying each one of these enzymes was able to synergistically hydrolyze filter paper at 55 °C, producing 20 μg glucose per mL cell suspension in 24 h.ConclusionWe could establish Pseudomonas putida as host for the surface display of cellulases, and provided proof-of-concept for a fast and simple cellulose breakdown process at elevated temperatures. This study opens up new perspectives for the application of P. putida in the production of biofuels and other biotechnological products.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0505-8) contains supplementary material, which is available to authorized users.

Biocatalytic behavior of a new Aspergillus niger whole-cell biocatalyst with high operational stability during the synthesis of green biosolvent isopropyl esters

Using microbial cell-surface displayed enzymes to produce chemicals is a promising green chemistry procedure, but few studies evaluated filamentous fungi cell-surface display systems. Here, a novel Aspergillus niger whole-cell biocatalyst with excellent operational stability was constructed using a heterologous anchor protein, and the details about its practical catalytic characteristics during the synthesis of green biosolvent isopropyl esters (IPEs) were investigated. Candida antarctica lipase B (CALB) was chosen as a model enzyme, fused to a Saccharomyces cerevisiae glycosylphosphatidylinositol protein, SED1, and displayed on the A. niger surface, the hydrolytic activity of the A. niger mycelium-surface displayed CALB reached 440.30U/g dry cell. In solvent-free esterifications of medium-long chain fatty acids and isopropanol, the A. niger/CALB-Flag-SED1 whole-cell biocatalyst showed great organic substrate tolerance, high thermostability, no significant lag time in a dry initial condition and no synthetic activity decrease after five batch reactions at 65°C. The highest yields of isopropyl laurate, isopropyl myristate, and isopropyl palmitate, 79.21, 81.62, and 81.41%, respectively, were obtained after a 6-h reaction. Acetone washing helped to remove the excessive water accumulating during esterification and benefitted the operational stability. The relative acid conversions of the three IPEs in batch 5 were 99.79, 99.10, and 96.83%, respectively, of those in batch 1. The A. niger/CALB-Flag-SED1 whole-cell biocatalyst is a promising alternative to commercial immobilized products, and SED1 can serve as an efficient anchor module to display industrial enzymes in an A. niger expression system.

Influence of saponins on the biodegradation of halogenated phenols

Biotransformation of aromatic compounds is a challenge due to their low aqueous solubility and sorptive losses. The main obstacle in this process is binding of organic pollutants to the microbial cell surface. To overcome these, we applied saponins from plant extract to the microbial culture, to increase pollutants solubility and enhance diffusive massive transfer. This study investigated the efficiency of Quillaja saponaria and Sapindus mukorossi saponins-rich extracts on biodegradation of halogenated phenols by Raoultella planticola WS2 and Pseudomonas sp. OS2, as an effect of cell surface modification of tested strains. Both strains display changes in inner membrane permeability and cell surface hydrophobicity in the presence of saponins during the process of halogenated phenols biotransformation. This allows them to more efficient pollutants removal from the environment. However, only in case of the Pseudomonas sp. OS2 the addition of surfactants to the culture improved effectiveness of bromo-, chloro- and fluorophenols biodegradation. Also introduction of surfactant allowed higher biodegradability of halogenated phenols and can shorten the process. Therefore this suggests that usage of plant saponins can indicate more successful halogenated phenols biodegradation for selected strains.

Radiation Damage and Racemic Protein Crystallography Reveal the Unique Structure of the GASA/Snakin Protein Superfamily.

Proteins from the GASA/snakin superfamily are common in plant proteomes and have diverse functions, including hormonal crosstalk, development, and defense. One 63-residue member of this family, snakin-1, an antimicrobial protein from potatoes, has previously been chemically synthesized in a fully active form. Herein the 1.5 Å structure of snakin-1, determined by a novel combination of racemic protein crystallization and radiation-damage-induced phasing (RIP), is reported. Racemic crystals of snakin-1 and quasi-racemic crystals incorporating an unnatural 4-iodophenylalanine residue were prepared from chemically synthesized d- and l-proteins. Breakage of the C-I bonds in the quasi-racemic crystals facilitated structure determination by RIP. The crystal structure reveals a unique protein fold with six disulfide crosslinks, presenting a distinct electrostatic surface that may target the protein to microbial cell surfaces.

Radiation Damage and Racemic Protein Crystallography Reveal the Unique Structure of the GASA/Snakin Protein Superfamily

AbstractProteins from the GASA/snakin superfamily are common in plant proteomes and have diverse functions, including hormonal crosstalk, development, and defense. One 63‐residue member of this family, snakin‐1, an antimicrobial protein from potatoes, has previously been chemically synthesized in a fully active form. Herein the 1.5 Å structure of snakin‐1, determined by a novel combination of racemic protein crystallization and radiation‐damage‐induced phasing (RIP), is reported. Racemic crystals of snakin‐1 and quasi‐racemic crystals incorporating an unnatural 4‐iodophenylalanine residue were prepared from chemically synthesized d‐ and l‐proteins. Breakage of the C−I bonds in the quasi‐racemic crystals facilitated structure determination by RIP. The crystal structure reveals a unique protein fold with six disulfide crosslinks, presenting a distinct electrostatic surface that may target the protein to microbial cell surfaces.

Binding of heavy metal ions in aggregates of microbial cells, EPS and biogenic iron minerals measured in-situ using metal- and glycoconjugates-specific fluorophores

Aggregates consisting of bacterial cells, extracellular polymeric substances (EPS) and Fe(III) minerals formed by Fe(II)-oxidizing bacteria are common at bulk or microscale chemical interfaces where Fe cycling occurs. The high sorption capacity and binding capacity of cells, EPS, and minerals controls the mobility and fate of heavy metals. However, it remains unclear to which of these component(s) the metals will bind in complex aggregates. To clarify this question, the present study focuses on 3D mapping of heavy metals sorbed to cells, glycoconjugates that comprise the majority of EPS constituents, and Fe(III) mineral aggregates formed by the phototrophic Fe(II)-oxidizing bacteria Rhodobacter ferrooxidans SW2 using confocal laser scanning microscopy (CLSM) in combination with metal- and glycoconjugates-specific fluorophores. The present study evaluated the influence of glycoconjugates, microbial cell surfaces, and (biogenic) Fe(III) minerals, and the availability of ferrous and ferric iron on heavy metal sorption. Analyses in this study provide detailed knowledge on the spatial distribution of metal ions in the aggregates at the sub-μm scale, which is essential to understand the underlying mechanisms of microbe–mineral–metal interactions. The heavy metals (Au3+, Cd2+, Cr3+, CrO42−, Cu2+, Hg2+, Ni2+, Pd2+, tributyltin (TBT) and Zn2+) were found mainly sorbed to cell surfaces, present within the glycoconjugates matrix, and bound to the mineral surfaces, but not incorporated into the biogenic Fe(III) minerals. Statistical analysis revealed that all ten heavy metals tested showed relatively similar sorption behavior that was affected by the presence of sorbed ferrous and ferric iron. Results in this study showed that in addition to the mineral surfaces, both bacterial cell surfaces and the glycoconjugates provided most of sorption sites for heavy metals. Simultaneously, ferrous and ferric iron ions competed with the heavy metals for sorption sites on the organic compounds. In summary, the information obtained by the present approach using a microbial model system provides important information to better understand the interactions between heavy metals and biofilms, and microbially formed Fe(III) minerals and heavy metals in complex natural environments.

Oral Vaccine Development by Molecular Display Methods Using Microbial Cells.

Oral vaccines are easier to administer than injectable vaccines. To induce an adequate immune response using vaccines, antigenic proteins are usually combined with adjuvant materials. This chapter presents methodologies for the design of oral vaccines using molecular display technology. In molecular display technology, antigenic proteins are displayed on a microbial cell surface with adjuvant ability. This technology would provide a quite convenient process to produce oral vaccines when the DNA sequence of an efficient antigenic protein is available. As an example, oral vaccines against candidiasis were introduced using two different molecular display systems with Saccharomyces cerevisiae and Lactobacillus casei.

Rapid acquisition of high-affinity DNA aptamer motifs recognizing microbial cell surfaces using polymer-enhanced capillary transient isotachophoresis.

We present a polymer-enhanced capillary transient isotachophoresis (PectI) selection methodology for acquisition of high-affinity (kinetically inert) DNA aptamers capable of recognizing distinct microbial cell surfaces, which requires only a single electrophoretic separation between particles (free cells and cells bound with aptamers) and molecules (unbound or dissociated DNA) in free solution.