Search

Aims Isolation, characterization and identification of possible microbial contaminant(s) in the inflated foil bag containing hop pellets packed and stored in a modified atmosphere. Methods and results Package gas of the inflated foil bag containing hop pellets was analysed by gas chromatography. Compared with the reference modified atmosphere, containing about 16 vol.% of CO2 , the inflated bag atmosphere contained 53 vol.% CO2 , suggesting possible microbial contamination. Therefore, several standard and mineral media, with added hop pellets or hop infusion, were used for cultivation at different temperatures under an anaerobic atmosphere. Cultivation in mineral medium with hop pellets yielded a bacterial isolate that was identified by MALDI-TOF mass spectrometry and verified by partial 16S rRNA gene analysis as Pantoea agglomerans, a known plant epiphyte. Conclusions A novel strain of P. agglomerans (designed as DBM 3696) was found to be suspicious of causing inflation of the foil bag containing dried hop pellets packed in modified atmosphere. Significance and impact of the study This study suggests that P. agglomerans, probably hop epiphyte, could cause sporadic inflation of bags with hop pellets packed in modified atmosphere causing logistical problems during bags transport.

Bioapatite ceramics produced from biogenic sources provide highly attractive materials for the preparation of artificial replacements since such materials are not only more easily accepted by living organisms, but bioapatite isolated from biowaste such as xenogeneous bones also provides a low-cost material. Nevertheless, the presence of organic compounds in the bioapatite may lead to a deterioration in its quality and may trigger an undesirable immune response. Therefore, procedures which ensure the elimination of organic compounds through bioapatite isolation are being subjected to intense investigation and the presence of remaining organic impurities is being determined through the application of various methods. Since current conclusions concerning the conditions suitable for the elimination of organic compounds remain ambiguous, we used the mass spectrometry-based proteomic approach in order to determine the presence of proteins or peptides in bioapatite samples treated under the most frequently employed conditions, i.e., the alkaline hydrothermal process and calcination at 500 °C. Since we also investigated the presence of proteins or peptides in treated bioapatite particles of differing sizes, we discovered that both calcination and the size of the bioapatite particles constitute the main factors influencing the presence of proteins or peptides in bioapatite. In fact, while intact proteins were detected even in calcinated bioapatite consisting of particles >250 µm, no proteins were detected in the same material consisting of particles

Enrichment of mortar mixtures by the addition of natural organic materials has a long history. Already in ancient times it has been empirically proved that proteinaceous additives significantly increase mortar durability, strength or plasticity. The knowledge of the protein occurrence in historical mortars would be very useful for the restoration work. However, some of historical recipes did not survive till these days or it is difficult to interpret them if they have survived, and, therefore,we are not able to prepare all kinds of mortars of an appropriate quality nowadays. The goal of this work was to identify and relatively quantify proteins contained in the model mortars samples enriched with four different protein additives (animal glue, blood, egg, and milk). For this purpose, aged mortar samples were analysed by LC-ESI-Q-TOF MS (Liquid Chromatography and ElectroSpray Ionization Quadrupole Time of Flight Mass Spectrometry) and the relative content of proteins was quantified by label-free technique. Number of semi-specific and non-specific peptides were detected in different ageing times and preferential sites in non-specific peptide were found. Principal component analysis (PCA) applied on MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization – Time of Flight) spectra was also used to differentiate between protein additives in mortars. Peptides that are stable in the prepared model mortars and characteristic for the individual protein additives were found and together with the results of relative quantification they confirmed the possibility of unambiguous identification of protein additives in century-old mortars that brings the opportunity to make an improved hypothesis about the old traditional recipes. By the mass spectrometric methods milk additive was identified in historical mortar from Swedish church dated back to 14th century.

The Mason-Pfizer monkey virus is a type D retrovirus, which assembles its immature particles in the cytoplasm prior to their transport to the host cell membrane. The association with the membrane is mediated by the N-terminally myristoylated matrix protein. To reveal the role of particular residues which are involved in the capsid-membrane interaction, covalent labelling of arginine, lysine and tyrosine residues of the Mason-Pfizer monkey virus matrix protein bound to artificial liposomes containing 95% of phosphatidylcholine and 5% phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2 ) was performed. The experimental results were interpreted by multiscale molecular dynamics simulations. The application of these two complementary approaches helped us to reveal that matrix protein specifically recognizes the PI(4,5)P2 molecule by the residues K20, K25, K27, K74, and Y28, while the residues K92 and K93 stabilizes the matrix protein orientation on the membrane by the interaction with another PI(4,5)P2 molecule. Residues K33, K39, K54, Y66, Y67, and K87 appear to be involved in the matrix protein oligomerization. All arginine residues remained accessible during the interaction with liposomes which indicates that they neither contribute to the interaction with membrane nor are involved in protein oligomerization. Proteins 2016; 84:1717-1727. © 2016 Wiley Periodicals, Inc.

Many ecological experiments are based on the extraction and downstream analyses of microorganisms from different environmental samples. Due to its high throughput, cost-effectiveness and rapid performance, Matrix Assisted Laser Desorption/Ionization Mass Spectrometry with Time-of-Flight detector (MALDI-TOF MS), which has been proposed as a promising tool for bacterial identification and classification, could be advantageously used for dereplication of recurrent bacterial isolates. In this study, we compared whole-cell MALDI-TOF MS-based analyses of 49 bacterial cultures to two well established bacterial identification and classification methods based on complete 16S rRNA gene sequence analyses: a phylotype-based approach, grounded on closest type strain assignment, and a sequence similarity-based approach involving a 98.65% sequence similarity threshold, which has been found to best delineate bacterial species. Culture classification using reference-based MALDI-TOF MS was comparable to that yielded by phylotype assignment up to the genus level. At the species level, agreement between 16S rRNA gene analysis and MALDI-TOF MS was found to be limited, potentially indicating that spectral reference databases need to be improved. We also evaluated the mass spectral similarity technique for species-level delineation which can be used independently of reference databases. We established optimal mass spectral similarity thresholds which group MALDI-TOF mass spectra of common environmental isolates analogically to phylotype- and sequence similarity-based approaches. When using a mass spectrum similarity approach, we recommend a mass range of 4 to 10 kDa for analysis, which is populated with stable mass signals and contains the majority of phylotype-determining peaks. We show that a cosine similarity (CS) threshold of 0.79 differentiate mass spectra analogously to 98.65% species-level delineation sequence similarity threshold, with corresponding precision and recall values of 0.70 and 0.73, respectively. When matched to species-level phylotype assignment, an optimal CS threshold of 0.92 was calculated, with associated precision and recall values of 0.83 and 0.64, respectively. Overall, our research indicates that a similarity-based MALDI-TOF MS approach can be routinely used for efficient dereplication of isolates for downstream analyses, with minimal loss of unique organisms. In addition, MALDI-TOF MS analysis has further improvement potential unlike 16S rRNA gene analysis, whose methodological limits have reached a plateau.

In the last decade, strains of the genera Franconibacter and Siccibacter have been misclassified as first Enterobacter and later Cronobacter . Because Cronobacter is a serious foodborne pathogen that affects premature neonates and elderly individuals, such misidentification may not only falsify epidemiological statistics but also lead to tests of powdered infant formula or other foods giving false results. Currently, the main ways of identifying Franconibacter and Siccibacter strains are by biochemical testing or by sequencing of the fusA gene as part of Cronobacter multilocus sequence typing (MLST), but in relation to these strains the former is generally highly difficult and unreliable while the latter remains expensive. To address this, we developed a fast, simple, and most importantly, reliable method for Franconibacter and Siccibacter identification based on intact-cell matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Our method integrates the following steps: data preprocessing using mMass software; principal-component analysis (PCA) for the selection of mass spectrum fingerprints of Franconibacter and Siccibacter strains; optimization of the Biotyper database settings for the creation of main spectrum projections (MSPs). This methodology enabled us to create an in-house MALDI MS database that extends the current MALDI Biotyper database by including Franconibacter and Siccibacter strains. Finally, we verified our approach using seven previously unclassified strains, all of which were correctly identified, thereby validating our method. IMPORTANCE We show that the majority of methods currently used for the identification of Franconibacter and Siccibacter bacteria are not able to properly distinguish these strains from those of Cronobacter . While sequencing of the fusA gene as part of Cronobacter MLST remains the most reliable such method, it is highly expensive and time-consuming. Here, we demonstrate a cost-effective and reliable alternative that correctly distinguishes between Franconibacter , Siccibacter , and Cronobacter bacteria and identifies Franconibacter and Siccibacter at the species level. Using intact-cell MALDI-TOF MS, we extend the current MALDI Biotyper database with 11 Franconibacter and Siccibacter MSPs. In addition, the use of our approach is likely to lead to a more reliable identification scheme for Franconibacter and Siccibacter strains and, consequently, a more trustworthy epidemiological picture of their involvement in disease.

Specific chemical modification is one of the basic techniques of protein chemistry. Inter alia can be used for detection of surface accessible amino acid residues; this information is of particular importance for studies of the participation of residues in intermolecular interactions of a protein. We achieved an improvement of the technique for arginine, aspartic, and glutamic acid modification using a simple combination of gel permeation and reversed-phase chromatography prior to mass spectrometry analysis. The improved protocol was tested on cytochrome c and M-PMV matrix protein. In both proteins, all accessible arginines and a high number of acidic amino acids were modified. These results indicate that the new protocol can be useful in protein structure analysis, generally.

RATIONALE The bacterial genus Cronobacter was established quite recently, in 2008. Therefore, its systematic classification is still in progress as well as the risk assessment of Cronobacter strains. The possibility of rapid identification within the biogroup level has an essential epidemiological significance. We examined the potential of mass spectrometry to accomplish this task on species Cronobacter sakazakii comprising eight different biogroups. METHODS Members of all Cronobacter sakazakii biogroups were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using intact cells. Analyses were performed on a Biflex IV MALDI-TOF mass spectrometer in the range of 2000 to 20 000 Da in linear mode with an accelerated voltage of 19 kV. RESULTS Optimal conditions for a proper identification of biogroups, such as suitable cultivation media or growth time of bacteria, were investigated. The biomarker patterns characterizing each of the Cronobacter sakazakii biogroups were obtained. The established identification protocol was applied to ten previously non-identified strains and their biogroups were successfully determined. CONCLUSIONS The presented work is the first report of successful and rapid bacterial biogroup taxonomy classification using MALDI-TOF-MS that could substitute demanding biochemical testing. Copyright © 2012 John Wiley & Sons, Ltd.

Matrix proteins play multiple roles both in early and late stages of the viral replication cycle. Their N-terminal myristoylation is important for interaction with the host cell membrane during virus budding. We used Escherichia coli, carrying N-myristoyltransferase gene, for the expression of the myristoylated His-tagged matrix protein of Mason-Pfizer monkey virus. An efficient, single-step purification procedure eliminating all contaminating proteins including, importantly, the non-myristoylated matrix protein was designed. The comparison of NMR spectra of matrix protein with its myristoylated form revealed substantial structural changes induced by this fatty acid modification.

Bacteria that are able to utilize biphenyl as a sole source of carbon were extracted and isolated from polychlorinated biphenyl (PCB)-contaminated soil vegetated by horseradish. Isolates were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The usage of MALDI Biotyper for the classification of isolates was evaluated and compared to 16S rRNA gene sequence analysis. A wide spectrum of bacteria was isolated, with Arthrobacter , Serratia , Rhodococcus , and Rhizobium being predominant. Arthrobacter isolates also represented the most diverse group. The use of MALDI Biotyper in many cases permitted the identification at the level of species, which was not achieved by 16S rRNA gene sequence analyses. However, some isolates had to be identified by 16S rRNA gene analyses if MALDI Biotyper-based identification was at the level of probable or not reliable identification, usually due to a lack of reference spectra included in the database. Overall, this study shows the possibility of using MALDI-TOF MS and MALDI Biotyper for the fast and relatively nonlaborious identification/classification of soil isolates. At the same time, it demonstrates the dominant role of employing 16S rRNA gene analyses for the identification of recently isolated strains that can later fill the gaps in the protein-based identification databases.