Comparison of detergent‐based sample preparation workflows for LTQ‐Orbitrap analysis of the Escherichia coli proteome

This work presents a comparative evaluation of several detergent‐based sample preparation workflows for the MS‐based analysis of bacterial proteomes, performed using the model organism Escherichia coli. Initially, RapiGest‐ and SDS‐based buffers were compared for their protein extraction efficiency and quality of the MS data generated. As a result, SDS performed best in terms of total protein yields and overall number of MS identifications, mainly due to a higher efficiency in extracting high molecular weight (MW) and membrane proteins, while RapiGest led to an enrichment in periplasmic and fimbrial proteins. Then, SDS extracts underwent five different MS sample preparation workflows, including: detergent removal by spin columns followed by in‐solution digestion (SC), protein precipitation followed by in‐solution digestion in ammonium bicarbonate or urea buffer, filter‐aided sample preparation (FASP), and 1DE separation followed by in‐gel digestion. On the whole, about 1000 proteins were identified upon LC‐MS/MS analysis of all preparations (>1100 with the SC workflow), with FASP producing more identified peptides and a higher mean sequence coverage. Each protocol exhibited specific behaviors in terms of MW, hydrophobicity, and subcellular localization distribution of the identified proteins; a comparative assessment of the different outputs is presented.     

Biofilm growth kinetics and nutrient (N/P) adsorption in an urban lake using reclaimed water: A quantitative baseline for ecological health assessment

Reclaimed wastewater reuse represents an effective method for partial resolution of increasing urban water shortages; however, reclaimed water may be characterized by significant contaminant loading, potentially affecting receiving ecosystem (and potentially human) health. The current study examined biofilm growth and nutrient adsorption in Olympic Lake (Beijing), the largest artificial urban lake in the world supplied exclusively by reclaimed wastewater. Findings indicate that solid particulate, extracellular polymeric substance (EPS) and metal oxide (Al, Fe, Mn) constituent masses adhere to a bacterial growth curve during biofilm formation and growth. Peak values were observed after ≈30 days, arrived at dynamic stability after ≈50 days and were affected by growth matrix surface roughness. These findings may be used to inform biofilm cultivation times for future biomonitoring. Increased growth matrix surface roughness (10.0 μm) was associated with more rapid biofilm growth and therefore an increased sensitivity to ecological variation in reclaimed water. Reclaimed water was found to significantly inhibit biofilm nutrient adsorption when compared with a “natural water” background, with elevated levels of metal oxides (Al, Fe, and Mn) and EPS representing the key substances actively influencing biofilm nutrient adsorption in reclaimed water. Results from the current study may be used to provide a quantitative baseline for future studies seeking to assess ecosystem health via monitoring of biofilms in the presence of reclaimed water through an improved quantitative understanding of biofilm kinetics in these conditions.     

Impact of Extraction Methods on the Detectable Protein Complement of Metaproteomic Analyses of Marine Sediments

Metaproteomic analysis targets proteins, the catalytic entities in the habitat, thereby providing direct insights into the metabolic activity of the community studied. A major challenge still remaining for metaproteomics is the effective and comprehensive extraction of proteins from environmental samples, due to their high complexity with respect to organismic diversity and abundance range. Moreover, in certain habitats, the inherent matrix may interfere with protein extraction. In recent years, several studies reported different protein extraction methods for soils known for their complex geochemistry, but only three analyzed marine sediments that generally comprise different though similarly complex geochemistry. In this study, the impact of four different extraction methods was investigated for coastal North Sea and deep sea Pacific Ocean sediments. The extraction methods comprised (i) phenol, (ii) SDS, (iii) a mixture of SDS and phenol, and (iv) urea and thiourea. Prior to extraction, a cell and protein standard (CPS) was added to the sediment samples to trace recovery of proteins from different subcellular locations as well as dissolved BSA. While each extraction method detected distinct peptide complements, SDS‐phenol extraction generally achieved highest protein yield and most comprehensive CPS protein identification. Application of two different methods was shown to further improve proteome coverage.     

Quantification of water and biomass in small colony variant PAO1 biofilms by confocal Raman microspectroscopy

The Raman spectra, water content, and biomass density of wild-type (WT) Pseudomonas aeruginosa PAO1, small colony variant (SCV) PAO1, and Pseudoalteromonas sp. NCIMB 2021 biofilms were compared in order to determine their variation with strain and species. Living, fully submerged biofilms were analyzed in situ by confocal Raman microspectroscopy for up to 2 weeks. Water to biomass ratios (W/BRs), which are the ratios of the O–H stretching vibration of water at 3,450 cm⁻¹ to the C–H stretching band characteristic of biomass at 2,950 cm⁻¹, were used to estimate the biomass density and cell density by comparison with W/BRs of protein solutions and bacterial suspensions, respectively, on calibration curves. The hydration within SCV biofilm colonies was extremely heterogeneous whereas W/BRs were generally constant in young WT biofilm colonies. The mean biomass in biofilm colonies of WT or colony cores of SCV was typically equivalent to 16% to 27% protein (w/v), but was 10% or less for NCIMB 2021. The corresponding cell densities were 7.5 to >10 × 10¹⁰ cfu mL⁻¹ for SCV, while the maximum cell density for NCIMB biofilms was 2.8 × 10¹⁰ cfu mL⁻¹.     

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.     

Comparisons of direct extraction methods of microbial DNA from different paddy soils

Molecular analyses for the study of soil microbial communities often depend on the direct extraction of DNA from soils. The present work compares the effectiveness of three different methods of extracting microbial DNA from seven different paddy soils. Comparison among different DNA extraction methods against different paddy soil samples revealed a marked variation in DNA yields from 3.18–20.17 μg DNA/g of dry soil. However, irrespective of the soil samples and extraction methods the DNA fragment size was >10 kb. Among the methods evaluated, method-C (chemical–enzymatic–mechanical) had better cell lysis efficiency and DNA yield. After purification of crude DNA by Purification Kit, A₂₆₀/A₂₃₀ and A₂₆₀/A₂₈₀ ratios of the DNA obtained by method-C reached up to 2.27 and 1.89, respectively, sustaining the efficacy of this technique in removing humic acid, protein and other contaminants. Results of the comprehensive evaluation of DNA extraction methods suggest that method-C is superior to other two methods (chemical–enzymatic and chemical–mechanical), and was the best choice for extraction of total DNA from soil samples. Since soil type and microbial community characteristics influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods according to experimental goals.     

Fungal surface protein mediated one-pot synthesis of stable and hemocompatible gold nanoparticles

Despite their large secretome and wide applications in bioprocesses, fungi remain underexplored in metal nanoparticles (MNP) biosynthesis. Previous studies have shown that cell surface proteins of Rhizopus oryzae play a crucial role in biomineralization of Au(III) to produce gold nanoparticles (AuNPs). Therefore, it is hypothesized that purified cell surface protein may produce in vitro AuNPs with narrow size distribution for biomedical and biocatalytic applications. However, different protein extraction methods might affect protein stability and the AuNP biosynthesis process. Herein, we have explored the extraction of cell surface proteins from R. oryzae using common detergents and reducing agent (sodium dodecyl sulfate (SDS) Triton X-100, and 1,4-dithiothreitol (DTT)) and their effect on the size and shape of the biosynthetic AuNPs. The surface proteins extracted with reducing agent (DTT) and non-ionic detergent (Triton X-100) produce spherical AuNPs with a mean particle size of 16 ± 7 nm, and 19 ± 4 nm, respectively, while the AuNPs produced by the surface protein extracted by ionic detergent (SDS) are flower-like AuNPs with broader size distribution of 43 ± 19 nm. This synthetic approach does not require use of any harsh chemicals, multistep preparation and separation process, favouring environmental sustainability. The biosynthetic AuNPs thus formed, are stable in different physiological buffers and hemocompatible, making them suitable for biomedical applications.     

Comparisons of protein extraction procedures and quantification methods for the proteomic analysis of Gram-positive <Emphasis Type="Italic">Paenibacillus</Emphasis> sp. strain D9

Four protein extraction methods and three protein quantification techniques were compared with Paenibacillus sp. whole cells. Proteins were extracted using conventional cell disruption techniques encompassing: sonication and glass bead vortexing, as well as BugBuster Master Mix extraction and Total Protein Kit extraction. The Bradford assay, Folin-Lowry assay and UV absorbance at 280 nm were used for protein quantification methods. Differences in protein profiles were examined by 2D-PAGE and subsequently analysed using PDQuest Advanced 2D Analysis software. All extraction methods revealed proteins over broad molecular weight range. UV absorbance at 280 nm using the NanoDrop™1000 and the Bradford assay yielded best quantification results. Rapid and effective disruption and quantification of Paenibacillus sp. strain D9 cells was successfully achieved using the combination of Total Protein Extraction Kit-UV280 followed by BugBuster Master-UV280.     

Updating the proteome of the uncultivable hemotrophic Mycoplasma suis in experimentally infected pigs

Mycoplasma suis belongs to the hemotrophic mycoplasmas that are associated with acute and chronic anemia in a wide range of livestock and wild animals. The inability to culture M. suis in vitro has hindered its characterization at the molecular level. Since the publication of M. suis genome sequences in 2011 only one proteome study has been published. Aim of the presented study was to significantly extend the proteome coverage of M. suis strain KI_3806 during acute infection by applying three different protein extraction methods followed by 1D SDS‐PAGE and LC‐MS/MS. A total of 404 of 795 M. suis KI_3806 proteins (50.8%) were identified. Data analysis revealed the expression of 83.7% of the predicted ORFs with assigned functions but also highlights the expression of 179 of 523 (34.2%) hypothetical proteins with unknown functions. Computational analyses identified expressed membrane‐associated hypothetical proteins that might be involved in adhesion or host–pathogen interaction. Furthermore, analyses of the expressed proteins indicated the existence of a hexose‐6‐phosphate‐transporter and an ECF transporter. In conclusion, our proteome study provides a further step toward the elucidation of the unique life cycle of M. suis and the establishment of an in vitro culture. All MS data have been deposited in the ProteomeXchange with identifier PXD002294 ( http://proteomecentral.proteomexchange.org/dataset/PXD002294 ).     

Production of β-galactosidase by Kluyveromyces marxianus MTCC 1388 using whey and effect of four different methods of enzyme extraction on β-galactosidase activity

Whey containing 4.4% (w/v) lactose was inoculated with Kluyveromyces marxianus MTCC 1389 for carrying out studies related to β-galactosidase production. β-galactosidase activity was found to be maximum after 30 h and further incubation resulted in decline in activity. The maximum cell biomass of 2.54 mg mL⁻¹ was observed after 36 h of incubation. Lactose concentration dropped drastically to 0.04 % from 4.40% after 36 h of incubation. Out of the four methods tested for extraction of enzyme, SDS — Chlorofom method was found to be best followed by Toluene — Acetone, sonication and homogenization with glass beads in that order. It could be concluded through this study that SDS — Chloroform is cheap and simple method for enzyme extraction from Kluyveromyces cells, which resulted in higher enzyme activity as compared to the activity observed using the remaining extraction methods. The study could also establish that whey could effectively be utilized for β-galactosidase production thus alleviating water pollution problems caused due to its disposal into the water streams.     

Proteolytic activity in enzymatic extracts from Carica papaya L. cv. Maradol harvest by-products

Proteolytic activity and the cysteine protease profile were determined for enzymatic extracts (EE) from Carica papaya L. cv. Maradol harvest by-products (stems, unripe fruit, petioles and leaves). The proportion of each by-product type in the sampled plantation was calculated. Polypeptide bands were identified by SDS–PAGE for each EE and molecular weight calculated for the cysteine proteases. Leaf and fruit tissue had the highest protein contents of the by-products. Leaf tissue also produced the highest total EE yield. All the SDS–PAGE gels for the EE’s exhibited an approximately 23 kDa band probably corresponding to papain. The zymography profiles of the EE’s were similar, with bands at approximately >202.8, 76.8, 55.4 and 46.5 kDa. The fruit EE had the highest specific proteolytic activity and the leaf EE the lowest. Fruit and stem by-products are the most promising for proteolytic enzyme extraction.     

Calcium-mediated Protein Folding and Stabilization of Salmonella Biofilm-associated Protein A

Biofilm-associated proteins (BAPs) are important for early biofilm formation (adhesion) by bacteria and are also found in mature biofilms. BapA from Salmonella is a ~ 386-kDa surface protein, comprising 27 tandem repeats predicted to be bacterial Ig-like (BIg) domains. Such tandem repeats are conserved for BAPs across different bacterial species, but the function of these domains is not completely understood. In this work, we report the first study of the mechanical stability of the BapA protein. Using magnetic tweezers, we show that the folding of BapA BIg domains requires calcium binding and the folded domains have differential mechanical stabilities. Importantly, we identify that > 100 nM concentration of calcium is needed for folding of the BIg domains, and the stability of the folded BIg domains is regulated by calcium over a wide concentration range from sub-micromolar (μM) to millimolar (mM). Only at mM calcium concentrations, as found in the extracellular environment, do the BIg domains have the saturated mechanical stability. BapA has been suggested to be involved in Salmonella invasion, and it is likely a crucial mechanical component of biofilms. Therefore, our results provide new insights into the potential roles of BapA as a structural maintenance component of Salmonella biofilm and also Salmonella invasion.     

The influence of cell immobilization by biofilm forming on the biodegradation capabilities of bacterial consortia

Recent biodegradation researches are focused on improving the deterioration efficiency and maintaining the stability of microbial activity during the process. Immobilizing bacterial cells by using the adhesive method is a simple way to achieve both objectives. The purpose of this study was to evaluate how the immobilization of bacterial cells by the biofilm forming method influences the biodegradation efficiency of two different bacterial consortia—low diesel oil degraders (base deterioration efficiency < 40%) and high phenol degraders (base deterioration efficiency > 90%). The degradation tests were carried out on four different carriers (expanded clay pellets, polypropylene, polyvinyl chloride rings and paperboard). The obtained results show that biofilms may considerably increase the efficiency of low degraders. The best diesel oil deterioration (80%) was achieved when paperboard was used as a carrier. However, the immobilization of high degraders did not significantly influence their base biodegradation potential. A 6% increase was noted only for the expanded clay pellets.     

The Effect of Ultrasonificated Extracts of <Emphasis Type="Italic">Spirulina maxima</Emphasis> on the Anticancer Activity

The effect of ultrasonic extraction on extraction yields, cytotoxicity, and anticancer activity of Spirulina maxima was investigated in this study. Optimal extraction conditions were determined as 60 kHz frequency at 60°C for 30 min with 120 W intensity, which resulted in 19.3% of extraction yields and 19.1% of cytotoxicity on normal human cells. Yields from conventional water and ethanol extraction were 15.8% at 100°C and 8.3% at 80°C, respectively. It was found that the extracts obtained by ultrasonic extraction process selectively inhibited the digestive-related cancer cell lines, such as human stomach cancer cells, having 89% of the highest inhibition ratio and 4.5 of the highest selectivity. In adding 0.5 mg/mL of the extract, human promyelocytic leukemia cells' cell differentiation was increased 1.72 times over that of the control. Expression level of B cell lymphoma-2 from Hep3B cell was also effectively suppressed by the extract obtained at 60 kHz and 60°C, leading to the inhibition of the early step of carcinogenesis. This work suggests that anticancer activity of the extracts is due to water-soluble polysaccharides rather than proteins and is further supported by the result that the ultrasonification extraction process can efficiently extract relatively intact polysaccharides rather than digesting the proteins in S. maxima by matrix assisted laser desorption ionization-time of flight and high performance size exclusion chromatography chromatogram analyses. Therefore, ultrasonic extraction increases both extraction yield and the biological activity of S. maxima extracts, which might be useful as an alternative natural anticancer agent in the medical and food industries.     

Metabolomics reveals stage-specific metabolic pathways of microbial communities in two-stage anaerobic fermentation of corn-stalk

Analysis of intracellular metabolites is essential to delineate metabolic pathways of microbial communities for evaluation and optimization of anaerobic fermentation processes. The metabolomics are reported for a microbial community during two stages of anaerobic fermentation of corn stalk in a biogas digester using GC–MS. Acetonitrile/methanol/water (2:2:1, by vol) was the best extraction solvent for microbial community analysis because it yielded the largest number of peaks (>200), the highest mean summed value of identified metabolites (23) and the best reproducibility with a coefficient of variation of 30 % among four different extraction methods. Inter-stage comparison of metabolite profiles showed increased levels of sugars and sugar alcohols during methanogenesis and fatty acids during acidogenesis. Identification of stage-specific metabolic pathways using metabolomics can therefore assist in monitoring and optimization of the microbial community for increased biogas production during anaerobic fermentation.     

Optimal method for efficiently removing extracellular nanofilaments from Shewanella oneidensis MR-1

The identification, production, and potential electron conductivity of bacterial extracellular nanofilaments is an area of great study, specifically in Shewanella oneidensis MR-1. While some studies focus on nanofilaments attached to the cellular body, many studies require the removal of these nanofilaments for downstream applications. The removal of nanofilaments from S. oneidensis MR-1 for further study requires not only that the nanofilaments be detached, but also for the cell bodies to remain intact. This is a study to both qualitatively (AFM) and quantitatively (LC/MS-MS) assess several nanofilament shearing methods and determine the optimal procedure. The best method for nanofilament removal, as judged by maximizing extracellular filamentous proteins and minimizing membrane and intracellular proteins, is vortexing a washed cell culture for 10 min.     

Characterisation of plant growth‐promoting rhizobacteria from rhizosphere soil of heat‐stressed and unstressed wheat and their use as bio‐inoculant

• High temperature induces several proteins in plants that enhance tolerance to high temperature shock. The fate of proteins synthesised in microbial cells or secreted into culture media by interacting microbes has not been fully elucidated. The present investigation aimed to characterise plant growth‐promoting rhizobacteria (PGPR) isolated from the rhizosphere of wheat genotypes (differing in tolerance to high temperature stress) and evaluate their performance as bioinoculant for use in wheat.
• Four bacterial strains, viz. Pseudomonas brassicacearum, Bacillus thuringiensis, Bacillus cereus strain W6 and Bacillus subtilis, were isolated from the rhizosphere of heat‐stressed and unstressed wheat genotypes. The wheat genotypes were exposed to high temperature stress at 45 °C for 10 days (3 h daily) at pre‐anthesis phase. Isolates were identified on the basis of morphology and biochemical characteristics, 16S rRNA gene sequencing and whole cell protein profiles. Results were further complemented by size exclusion chromatography (SEC) with fast protein liquid chromatography (FPLC) and SDS PAGE of 80% ammonium sulphate precipitates of the cell‐free supernatants.
• Isolates were positive for catalase, oxidases and antimicrobial activity . P. brassicacearum from the rhizosphere of the heat‐tolerant genotype was more efficient in phosphate solubilisation, bacteriocin production, antifungal and antibacterial activity against Helminthosporium sativum, Fusarium moniliforme and Klebsiella pneumonia, respectively. The inoculated seedlings had significantly higher root and shoot fresh weight, enhanced activity of antioxidant enzymes, proline and protein content. Total profiling of the culture with SDS‐PAGE indicated expression of new protein bands in 95 kDa in P. brassicacearum.
• Temperature‐induced changes in PGPR isolates are similar to those in the host plant. P. brassicacearum may be a good candidate for use in biofertiliser production for plants exposed to high temperature stress.

     

Extraction of extracellular polymeric substances from extreme acidic microbial biofilms

The efficiency of five extraction methods for extracellular polymeric substances (EPS) was compared on three benthic eukaryotic biofilms isolated from an extreme acidic river, Río Tinto (SW, Spain). Three chemical methods (MilliQ water, NaCl, and ethylenediamine tetraacetic acid [EDTA]) and two physical methods (Dowex 50.8 and Crown Ether cation exchange resins) were tested. The quality and quantity of the EPS extracted from acidic biofilms varied according to which EPS extraction protocol was used. Higher amounts were obtained when NaCl and Crown Ether resins were used as extractant agents, followed by EDTA, Dowex, and MilliQ. EPS amounts varied from approximately 155 to 478 mg g⁻¹ of dry weight depending on the extraction method and biofilm analyzed. EPS were primarily composed of carbohydrate, heavy metals, and humic acid, plus small quantities of proteins and DNA. Neutral hexose concentrations corresponded to more than 90% of the total EPS dry weight. The proportions of each metals in the EPS extracted with EDTA are similar to the proportions present in the water from each locality where the biofilms were collected except for Al, Cu, Zn, and Pb. In this study, the extracellular matrix heavy metal sorption efficiencies of five methods for extracting EPS from eukaryotic acidic biofilms were compared.     

Comparison of five commercial extraction kits for subsequent membrane protein profiling

Membrane proteins account for 70–80% of all pharmaceutical targets emphasizing their clinical relevance. Identification of new, differentially expressed membrane proteins reflecting distinct disease properties is thus of high importance. Unfortunately, isolation and analysis of membrane-bound proteins is hampered by their relative low abundance in total cell lysates, their frequently large size and their hydrophobic properties. We thus aimed to identify protocols that allow for highly efficient isolation and purification of membrane-bound proteins for subsequent protein profiling. We present a comparative study of different membrane protein extraction methods that vary in total protein yield between 0.02 and 4.8 mg using constant cell pellets of the colorectal carcinoma cell line SW620. We also demonstrate by means of polyacrylamide gel electrophoresis (SDS–PAGE) and Western blot analysis that the majority of commercial membrane extraction kits harbor a substantial cytosolic contamination of their membranous fraction. Based on purity of membranous fraction, protein yield, time and costs, we show superiority of two commercial extraction kits for downstream proteome analyses of membrane proteins.