Meltome atlas-thermal proteome stability across the tree of life.

We have used a mass spectrometry-based proteomic approach to compile an atlas of the thermal stability of 48,000 proteins across 13 species ranging from archaea to humans and covering melting temperatures of 30-90 °C. Protein sequence, composition and size affect thermal stability in prokaryotes and eukaryotic proteins show a nonlinear relationship between the degree of disordered protein structure and thermal stability. The data indicate that evolutionary conservation of protein complexes is reflected by similar thermal stability of their proteins, and we show examples in which genomic alterations can affect thermal stability. Proteins of the respiratory chain were found to be very stable in many organisms, and human mitochondria showed close to normal respiration at 46 °C. We also noted cell-type-specific effects that can affect protein stability or the efficacy of drugs. This meltome atlas broadly defines the proteome amenable to thermal profiling in biology and drug discovery and can be explored online at http://meltomeatlas.proteomics.wzw.tum.de:5003/ and http://www.proteomicsdb.org.

Identification of Bulgarian Sourdough Microbiota by Metagenomic Approach Using Three Commercially Available DNA Extraction Protocols.

Research background: Sourdough is a spontaneously formed, complex microbial ecosystem of various lactic acid bacteria (LAB) and yeast which, by producing specific metabolites, determines the quality of the baked products. In order to design and control the sourdough with preferred nutritional characteristics, it is crucial that the LAB diversity of the product of interest be elucidated. Experimental approach: Using the opportunities of next-generation sequencing (NGS) of the V1-V3 hypervariable gene region of 16S rRNA, we studied the microbial ecosystem of a whole grain sourdough made of Triticum monococcum, originating from Southwestern Bulgaria. Since the DNA extraction method is considered crucial for the accuracy of the sequencing results, as it can introduce significant differences in the examined microbiota, we used three different commercial kits for DNA isolation and analyzed their impact on the observed bacterial diversity. Results and conclusions: All three DNA extraction kits provided bacterial DNA which passed quality control and was successfully sequenced on Illumina MiSeq platform. The results received from the different DNA protocols showed variations in the microbial profiles. Alpha diversity indices (ACE, Chao1, Shannon, and Simpson) were also different among the three groups of results. Nevertheless, a strong dominance of phylum Firmicutes, class Bacilli, order Lactobacillales, represented mostly by family Lactobacillaceae, genus Lactobacillus (relative abundance of 63.11-82.28%) and family Leuconostocaceae, genus Weissella (relative abundance of 3.67-36.31%) was observed. Lactiplantibacillus plantarum and Levilactobacillus brevis with relative abundance of 16.15-31.24% and 6.21-16.29% respectively, were the two dominant species identified in all three DNA isolates. Novelty and scientific contribution: The presented results give insight into the taxonomic composition of bacterial community of a specific Bulgarian sourdough. Having in mind that the sourdough is a difficult matrix for DNA isolation on the one hand, and that there is no standardized DNA extraction protocol for this matrix on the other hand, this pilot study aims to give a small contribution to the future establishment and validation of such a protocol, which will allow accurate assessment of the specific microbiota of sourdough samples.

An Unprecedented Tolerance to Deletion of the Periplasmic Chaperones SurA, Skp, and DegP in the Nosocomial Pathogen Acinetobacter baumannii.

The outer membrane (OM) of Gram-negative bacteria efficiently protects from harmful environmental stresses such as antibiotics, disinfectants, or dryness. The main constituents of the OM are integral OM ß-barrel proteins (OMPs). In Gram-negative bacteria such as Escherichia coli, Yersinia enterocolitica, and Pseudomonas aeruginosa, the insertion of OMPs depends on a sophisticated biogenesis pathway. This comprises the SecYEG translocon, which enables inner membrane (IM) passage; the chaperones SurA, Skp, and DegP, which facilitate the passage of ß-barrel OMPs through the periplasm; and the ß-barrel assembly machinery (BAM), which facilitates insertion into the OM. In E. coli, Y. enterocolitica, and P. aeruginosa, the deletion of SurA is particularly detrimental and leads to a loss of OM integrity, sensitization to antibiotic treatment, and reduced virulence. In search of targets that could be exploited to develop compounds that interfere with OM integrity in Acinetobacter baumannii, we employed the multidrug-resistant strain AB5075 to generate single gene knockout strains lacking individual periplasmic chaperones. In contrast to E. coli, Y. enterocolitica, and P. aeruginosa, AB5075 tolerates the lack of SurA, Skp, or DegP with only weak mutant phenotypes. While the double knockout strains ΔsurAΔskp and ΔsurAΔdegP are conditionally lethal in E. coli, all double deletions were well tolerated by AB5075. Strikingly, even a triple-knockout strain of AB5075, lacking surA, skp, and degP, was viable. IMPORTANCE Acinetobacter baumannii is a major threat to human health due to its ability to persist in the hospital environment, resistance to antibiotic treatment, and ability to deploy multiple and redundant virulence factors. In a rising number of cases, infections with multidrug-resistant A. baumannii end up fatally, because all antibiotic treatment options fail. Thus, novel targets have to be identified and alternative therapeutics have to be developed. The knockout of periplasmic chaperones has previously proven to significantly reduce virulence and even break antibiotic resistance in other Gram-negative pathogens. Our study in A. baumannii demonstrates how variable the importance of the periplasmic chaperones SurA, Skp, and DegP can be and suggests the existence of mechanisms allowing A. baumannii to cope with the lack of the three periplasmic chaperones.

Towards valorization of pectin-rich agro-industrial residues: Engineering of Saccharomyces cerevisiae for co-fermentation of d-galacturonic acid and glycerol.

Pectin-rich plant biomass residues represent underutilized feedstocks for industrial biotechnology. The conversion of the oxidized monomer d-galacturonic acid (d-GalUA) to highly reduced fermentation products such as alcohols is impossible due to the lack of electrons. The reduced compound glycerol has therefore been considered an optimal co-substrate, and a cell factory able to efficiently co-ferment these two carbon sources is in demand. Here, we inserted the fungal d-GalUA pathway in a strain of the yeast S. cerevisiae previously equipped with an NAD-dependent glycerol catabolic pathway. The constructed strain was able to consume d-GalUA with the highest reported maximum specific rate of 0.23 g gCDW-1 h-1 in synthetic minimal medium when glycerol was added. By means of a 13C isotope-labelling analysis, carbon from both substrates was shown to end up in pyruvate. The study delivers the proof of concept for a co-fermentation of the two 'respiratory' carbon sources to ethanol and demonstrates a fast and complete consumption of d-GalUA in crude sugar beet pulp hydrolysate under aerobic conditions. The future challenge will be to achieve co-fermentation under industrial, quasi-anaerobic conditions.

A rapid and efficient strategy to identify and recover biosynthetic gene clusters from soil metagenomes.

Culture-independent metagenomic approaches offer a promising solution to the discovery of therapeutically relevant compounds such as antibiotics by enabling access to the hidden biosynthetic potential of microorganisms. These strategies, however, often entail laborious, multi-step, and time-consuming procedures to recover the biosynthetic gene clusters (BGCs) from soil metagenomes for subsequent heterologous expression. Here, we developed an efficient method we called single Nanopore read cluster mining (SNRCM), which enables the fast recovery of complete BGCs from a soil metagenome using long- and short-read sequencing. A metagenomic fosmid library of 83,700 clones was generated and sequenced using Nanopore as well as Illumina technologies. Hybrid assembled contigs of the sequenced fosmid library were subsequently analyzed to identify BGCs encoding secondary metabolites. Using SNRCM, we aligned the identified BGCs directly to Nanopore long-reads and were able to detect complete BGCs on single fosmids. This enabled us to select for and recover BGCs of interest for subsequent heterologous expression attempts. Additionally, the sequencing data of the fosmid library and its corresponding metagenomic DNA enabled us to assemble and recover a large nonribosomal peptide synthetase (NRPS) BGC from three different fosmids of our library and to directly amplify and recover a complete lasso peptide BGC from the high-quality metagenomic DNA. Overall, the strategies presented here provide a useful tool for accelerating and facilitating the identification and production of potentially interesting bioactive compounds from soil metagenomes. KEY POINTS: • An efficient approach for the recovery of BGCs from soil metagenomes was developed to facilitate natural product discovery. • A fosmid library was constructed from soil metagenomic HMW DNA and sequenced via Illumina and Nanopore. • Nanopore long-reads enabled the direct identification and recovery of complete BGCs on single fosmids.

Identification of Drug Resistance Determinants in a Clinical Isolate of Pseudomonas aeruginosa by High-Density Transposon Mutagenesis.

With the aim to identify potential new targets to restore antimicrobial susceptibility of multidrug-resistant (MDR) Pseudomonas aeruginosa isolates, we generated a high-density transposon (Tn) insertion mutant library in an MDR P. aeruginosa bloodstream isolate (isolate ID40). The depletion of Tn insertion mutants upon exposure to cefepime or meropenem was measured in order to determine the common resistome for these clinically important antipseudomonal ß-lactam antibiotics. The approach was validated by clean deletions of genes involved in peptidoglycan synthesis/recycling, such as the genes for the lytic transglycosylase MltG, the murein (Mur) endopeptidase MepM1, the MurNAc/GlcNAc kinase AmgK, and the uncharacterized protein YgfB, all of which were identified in our screen as playing a decisive role in survival after treatment with cefepime or meropenem. We found that the antibiotic resistance of P. aeruginosa can be overcome by targeting usually nonessential genes that turn essential in the presence of therapeutic concentrations of antibiotics. For all validated genes, we demonstrated that their deletion leads to the reduction of ampC expression, resulting in a significant decrease in ß-lactamase activity, and consequently, these mutants partly or completely lost resistance against cephalosporins, carbapenems, and acylaminopenicillins. In summary, the determined resistome may comprise promising targets for the development of drugs that may be used to restore sensitivity to existing antibiotics, specifically in MDR strains of P. aeruginosa.

Oats as a matrix of choice for developing fermented functional beverages.

Development of oat-based fermented beverages started in Europe in the past 30 years with the rise of the functional foods market. It is based on the increasing consumer demand for health drinks and value added foods and on the scientific insights on the nutrition composition of oats. The main health effects of oats are attributed to their high ß-glucan content, which is proved to lower blood cholesterol and the intestinal absorption of glucose thus preventing diseases like cardiovascular injury, dyslipidemia, hypertension, inflammatory state and diabetes type 2. Another important role of ß- glucan is its prebiotic function in the gastrointestinal tract, supporting the growth of beneficial microbial groups. The slowly digestible fraction of oat starch has a functional role as it moderates the glycaemic response. Oats are also a valuable source of highquality proteins, unsaturated lipids and antioxidants. In addition, oats are appropriate for people suffering from celiac disease because they do not contain gluten. Oat grain processing involves several steps, including thermal processes aiming to prevent oat products from rapid enzymatic deterioration and ensure storage stability. Oat drinks are formulated through processing oat with a liquid ingredient. Further, this matrix is inoculated with lactic acid bacteria to produce a fermented beverage. In some, drinks, probiotic lactic acid bacteria were used to increase product functional value. Thus, the ancient concept of cereal-based fermented foods is implemented into development of new functional oat-based fermented beverages and several products are already marketed in Europe as healthy, fast and convenient supplementary foods.

Structural basis for cellulose binding by the type A carbohydrate-binding module 64 of Spirochaeta thermophila.

Spirochaeta thermophila secretes seven glycoside hydrolases for plant biomass degradation that carry a carbohydrate-binding module 64 (CBM64) appended at the C-terminus. CBM64 adsorbs to various ß1-4-linked pyranose substrates and shows high affinity for cellulose. We present the first crystal structure of a CBM64 at 1.2 Å resolution, which reveals a jelly-roll-like fold corresponding to a surface-binding type A CBM. Modeling of its interaction with cellulose indicates that CBM64 achieves association with the hydrophobic face of ß-linked pyranose chains via a unique coplanar arrangement of four exposed tryptophan side chains. Proteins 2016; 84:855-858. © 2016 Wiley Periodicals, Inc.

Characterization of chromosomal and megaplasmid partitioning loci in Thermus thermophilus HB27.

BACKGROUND: In low-copy-number plasmids, the partitioning loci (par) act to ensure proper plasmid segregation and copy number maintenance in the daughter cells. In many bacterial species, par gene homologues are encoded on the chromosome, but their function is much less understood. In the two-replicon, polyploid genome of the hyperthermophilic bacterium Thermus thermophilus, both the chromosome and the megaplasmid encode par gene homologues (parABc and parABm, respectively). The mode of partitioning of the two replicons and the role of the two Par systems in the replication, segregation and maintenance of the genome copies are completely unknown in this organism. RESULTS: We generated a series of chromosomal and megaplasmid par mutants and sGFP reporter strains and analyzed them with respect to DNA segregation defects, genome copy number and replication origin localization. We show that the two ParB proteins specifically bind their cognate centromere-like sequences parS, and that both ParB-parS complexes localize at the cell poles. Deletion of the chromosomal parAB genes did not apparently affect the cell growth, the frequency of cells with aberrant nucleoids, or the chromosome and megaplasmid replication. In contrast, deletion of the megaplasmid parAB operon or of the parB gene was not possible, indicating essentiality of the megaplasmid-encoded Par system. A mutant expressing lower amounts of ParABm showed growth defects, a high frequency of cells with irregular nucleoids and a loss of a large portion of the megaplasmid. The truncated megaplasmid could not be partitioned appropriately, as interlinked megaplasmid molecules (catenenes) could be detected, and the ParBm-parSm complexes in this mutant lost their polar localization. CONCLUSIONS: We show that in T. thermophilus the chromosomal par locus is not required for either the chromosomal or megaplasmid bulk DNA replication and segregation. In contrast, the megaplasmid Par system of T. thermophilus is needed for the proper replication and segregation of the megaplasmid, and is essential for its maintenance. The two Par sets in T. thermophilus appear to function in a replicon-specific manner. To our knowledge, this is the first analysis of Par systems in a polyploid bacterium.

Process engineering for bioflavour production with metabolically active yeasts - a mini-review.

Flavours are biologically active molecules of large commercial interest in the food, cosmetics, detergent and pharmaceutical industries. The production of flavours can take place by either extraction from plant materials, chemical synthesis, biological conversion of precursor molecules or de novo biosynthesis. The latter alternatives are gaining importance through the rapidly growing fields of systems biology and metabolic engineering, giving efficient production hosts for the so-called 'bioflavours', which are natural flavour and/or fragrance compounds obtained with cell factories or enzymatic systems. Yeasts are potential production hosts for bioflavours. In this mini-review, we give an overview of bioflavour production in yeasts from the process-engineering perspective. Two specific examples, production of 2-phenylethanol and vanillin, are used to illustrate the process challenges and strategies used.

Identification of ecotype-specific marker genes for categorization of beer-spoiling Lactobacillus brevis.

The tolerance to hop compounds, which is mainly associated with inhibition of bacterial growth in beer, is a multi-factorial trait. Any approaches to predict the physiological differences between beer-spoiling and non-spoiling strains on the basis of a single marker gene are limited. We identified ecotype-specific genes related to the ability to grow in Pilsner beer via comparative genome sequencing. The genome sequences of four different strains of Lactobacillus brevis were compared, including newly established genomes of two highly hop tolerant beer isolates, one strain isolated from faeces and one published genome of a silage isolate. Gene fragments exclusively occurring in beer-spoiling strains as well as sequences only occurring in non-spoiling strains were identified. Comparative genomic arrays were established and hybridized with a set of L. brevis strains, which are characterized by their ability to spoil beer. As result, a set of 33 and 4 oligonucleotide probes could be established specifically detecting beer-spoilers and non-spoilers, respectively. The detection of more than one of these marker sequences according to a genetic barcode enables scoring of L. brevis for their beer-spoiling potential and can thus assist in risk evaluation in brewing industry.

Time lag model for batch bioreactor simulation accounting the effect of micro-organism mortality.

In the present work, a generalization of the classical model of Monod accounting the influence of both delayed and instant mortalities on the dynamics of the micro-organism population is proposed. The model was analysed and compared with respect to its quality and applicability for simulation of the cultivation process of micro-organisms. Existence of a unique global positive solution of the Cauchy problem for the proposed model is proved and explicit relations between the decay parameters and the nutrition substrate concentration are obtained. These mathematical results allow us to calculate the nutrient substrate concentration which guarantees that the biomass concentration is maximal for every specific type of taxonomic groups of micro-organisms (bacteria, yeasts).

Epidemiology of fragile X syndrome: a systematic review and meta-analysis.

Prevalence estimates for fragile X syndrome vary considerably. This systematic review and meta-analysis was conducted to provide an accurate prevalence estimate for this disorder using primary publications in PubMed, Embase, and the Cochrane library. Data were pooled using Bayesian fixed-effects and random-effects models. Primary analyses assessed the frequency of the full mutation and premutation in males and females in the total population (no bias against individuals with intellectual disability) and in female carriers of the premutation in normal populations (biased against individuals with intellectual disability), based on diagnosis by polymerase chain reaction or Southern blotting. A sensitivity analysis included studies using any diagnostic testing method and conference abstracts. Sixty-eight recorded observations provided data for the primary (56 observations) and sensitivity (12 observations) analysis. Using the random-effects model, frequency of the full mutation was 1.4 (95% CI: 0.1-3.1) per 10,000 males and 0.9 (95% CI: 0.0-2.9) per 10,000 females (1:7,143 and 1:11,111, respectively) in the total population. The premutation frequency was 11.7 (95% CI: 6.0-18.7) per 10,000 males and 34.4 (95% CI: 6.3-83.3) per 10,000 for females (1:855 and 1:291, respectively) in the total population. The prevalence of female carriers of the premutation in the normal population was 34.4 (95% CI: 8.9-60.3) per 10,000, or 1:291. Sensitivity analyses resulted in similar prevalence estimates but with wider heterogeneity. Prevalence estimates for the full mutation from this meta-analysis are lower than those in previous reviews of fragile X syndrome epidemiological data.

Alternative hosts for functional (meta)genome analysis.

Microorganisms are ubiquitous on earth, often forming complex microbial communities in numerous different habitats. Most of these organisms cannot be readily cultivated in the laboratory using standard media and growth conditions. However, it is possible to gain access to the vast genetic, enzymatic, and metabolic diversity present in these microbial communities using cultivation-independent approaches such as sequence- or function-based metagenomics. Function-based analysis is dependent on heterologous expression of metagenomic libraries in a genetically amenable cloning and expression host. To date, Escherichia coli is used in most cases; however, this has the drawback that many genes from heterologous genomes and complex metagenomes are expressed in E. coli either at very low levels or not at all. This review emphasizes the importance of establishing alternative microbial expression systems consisting of different genera and species as well as customized strains and vectors optimized for heterologous expression of membrane proteins, multigene clusters encoding protein complexes or entire metabolic pathways. The use of alternative host-vector systems will complement current metagenomic screening efforts and expand the yield of novel biocatalysts, metabolic pathways, and useful metabolites to be identified from environmental samples.

Proteomic analysis of Viscozyme L and its major enzyme components for pectic substrate degradation.

Enzymatic degradation of plant biomass requires the coordinated action of various enzymes. In this study, the production of reducing sugars from pectic substrates and sugar beet pulp (SBP) was investigated and compared using commercial enzyme preparations, including M2, pectinase (E1), Viscozyme L (V-L) and L-40. V-L, a cellulolytic enzyme mix produced by Aspergillus sp. was further evaluated as the most robust enzyme cocktail with the strongest SBP degradation ability in terms of the release of monosaccharides, methanol, and acetate from SBP. Mass-spectrometry-based proteomics analysis of V-L revealed 156 individual proteins. Of these, 101 proteins were annotated as containing a carbohydrate-active enzyme module. Notably, of the 50 most abundant proteins, ca. 44 % were predicted to be involved in pectin degradation. To reveal the role of individual putative key enzymes in pectic substrate decomposition, two abundant galacturonases (PglA and PglB), were heterologously expressed in Pichia pastoris and further characterized. PglA and PglB demonstrated maximum activity at 57 °C and 68 °C, respectively, and exhibited endo-type cleavage patterns towards polygalacturonic acid. Further studies along this line may lead to a better understanding of efficient SBP degradation and may help to design improved artificial enzyme mixtures with lower complexity for future application in biotechnology.

Impact of resistant starch: Absorption of dietary minerals, glycemic index and oxidative stress in healthy rats.

BACKGROUND & AIMS: Resistant starch (RS) is a prebiotic fiber that has been scientifically shown to control the development of obesity. Prebiotic role of RS has also seen to be very important as it helps gut bacteria to regulate fermentation and fatty acid production. This study aimed to check the different levels of RS on glycemic index, oxidative stress and mineral absorption rate in healthy rat models. To evaluate these objectives, the trial was conducted for 40 days of follow up; 10 days were the adjustment period and the collection period over 30 days. METHODS: Thirty-six healthy female Wistar rats were divided into 4 groups of (9 animals each) NC (Normal Control: without resistant starch), RS0.20 (resistant starch: 0.20 g/kg body weight), RS0.30 (resistant starch: 0.30 g/kg body weight), RS0.40 (resistant starch: 0.40 g/kg body weight). All the diets were isocaloric and isonitroginous. RESULTS: The impact of different levels of RS on the dry-matter intake (DMI) presented statistically significant results (p ≤ 0.05): DMI was reduced in RS (0.02) fed rats as compared to NC rats in first 3 weeks; and after 4th and 5th weeks, there was a DMI reduction of 28% in RS (0.04) fed rats. Moreover, there was no significant increase in the nutrient intake in all RS diets. The dry-matter (DM) digestibility was statistically significantly (P ≤ 0·05), which increased in all rats fed with different level of RS. The weight loss showed statistically significant results: RS (0.04) exhibited 19 g reduction in weight as compared with NC rats. Significant increase was observed in total oxidant status (TOS), in all the RS fed rats when compared with NC rats. The levels of Mg, Ca, Fe and Zn were shown to be decrease in feces analysis, which proves their better absorbance in gut. Statistically significant increase was observed in antioxidant capacity, whereas significant decrease was observed in the total weight of the animals, showing the role of RS in controlling obesity. CONCLUSIONS: Overall, significant results were found in all dosage level of RS but long term administration of the higher dosage level (RS0.40) may need to be studied for enhanced results. RS can help improve insulin sensitivity in overweight adults.

Viral genome sequencing to decipher in-hospital SARS-CoV-2 transmission events.

The SARS-CoV-2 pandemic has highlighted the need to better define in-hospital transmissions, a need that extends to all other common infectious diseases encountered in clinical settings. To evaluate how whole viral genome sequencing can contribute to deciphering nosocomial SARS-CoV-2 transmission 926 SARS-CoV-2 viral genomes from 622 staff members and patients were collected between February 2020 and January 2021 at a university hospital in Munich, Germany, and analysed along with the place of work, duration of hospital stay, and ward transfers. Bioinformatically defined transmission clusters inferred from viral genome sequencing were compared to those inferred from interview-based contact tracing. An additional dataset collected at the same time at another university hospital in the same city was used to account for multiple independent introductions. Clustering analysis of 619 viral genomes generated 19 clusters ranging from 3 to 31 individuals. Sequencing-based transmission clusters showed little overlap with those based on contact tracing data. The viral genomes were significantly more closely related to each other than comparable genomes collected simultaneously at other hospitals in the same city (n = 829), suggesting nosocomial transmission. Longitudinal sampling from individual patients suggested possible cross-infection events during the hospital stay in 19.2% of individuals (14 of 73 individuals). Clustering analysis of SARS-CoV-2 whole genome sequences can reveal cryptic transmission events missed by classical, interview-based contact tracing, helping to decipher in-hospital transmissions. These results, in line with other studies, advocate for viral genome sequencing as a pathogen transmission surveillance tool in hospitals.

Screening and expression of genes from metagenomes.

Microorganisms are the most abundant and widely spread organisms on earth. They colonize a huge variety of natural and anthropogenic environments, including very specialized ecological niches and even extreme habitats, which are made possible by the immense metabolic diversity and genetic adaptability of microbes. As most of the organisms from environmental samples defy cultivation, cultivation-independent metagenomics approaches have been applied since more than one decade to access and characterize the phylogenetic diversity in microbial communities as well as their metabolic potential and ecological functions. Thereby, metagenomics has fully emerged as an own scientific field for mining new biocatalysts for many industrially relevant processes in biotechnology and pharmaceutics. This review summarizes common metagenomic approaches ranging from sampling, isolation of nucleic acids, construction of metagenomic libraries and their evaluation. Sequence-based screenings implement next-generation sequencing platforms, microarrays or PCR-based methods, while function-based analysis covers heterologous expression of metagenomic libraries in diverse screening setups. Major constraints and advantages of each strategy are described. The importance of alternative host-vector systems is discussed, and in order to underline the role of phylogenetic and physiological distance from the gene donor and the expression host employed, a case study is presented that describes the screening of a genomic library from an extreme thermophilic bacterium in both Escherichia coli and Thermus thermophilus. Metatranscriptomics, metaproteomics and single-cell-based methods are expected to complement metagenomic screening efforts to identify novel biocatalysts from environmental samples.

Comparative direct infusion ion mobility mass spectrometry profiling of Thermus thermophilus wild-type and mutant ∆cruC carotenoid extracts.

The major carotenoid species isolated from the thermophilic bacterium Thermus thermophilus HB27 have been identified as zeaxanthin-glucoside-fatty acid esters (thermozeaxanthins and thermobiszeaxanthins). Most of the genes of the proposed T. thermophilus carotenoid pathway could be found in the genome, but there is less clarity about the genes which encode the enzymes performing the final carotenoid glycosylation and acylation steps. To get a further insight into the biosynthesis of thermo(bis)zeaxanthins in T. thermophilus, we deleted the megaplasmid open reading frame TT_P0062 (termed cruC) by both exchanging it with a kanamycin resistance cassette (ΔcruC:kat) and by generating a markerless gene deletion strain (ΔcruC). A fast and efficient electrospray ionization-ion mobility-time-of-flight mass spectrometry method via direct infusion was developed to compare the carotenoid profiles of wild type and mutant T. thermophilus cell culture extracts. These comparisons revealed significant alterations in the carotenoid composition of the ΔcruC mutant, which was found to accumulate zeaxanthin. This is the first experimental evidence that the ORF encodes the glycosyltransferase enzyme necessary for the glycosylation of zeaxanthin in the final modification steps of the thermozeaxanthin biosynthesis in T. thermophilus HB27. Also, the proposed method for direct determination of carotenoid amounts and species in crude acetone extracts represents an improvement over existing methods in terms of speed and sensitivity and may be applicable in high-throughput analyses of other terpenoids as well as other important bacterial metabolites like fatty acids and their derivatives.

On the Molecular Selection of Exopolysaccharide-Producing Lactic Acid Bacteria from Indigenous Fermented Plant-Based Foods and Further Fine Chemical Characterization.

Exopolysaccharides (EPSs) produced by lactic acid bacteria present a particular interest for the food industry since they can be incorporated in foods via in situ production by selected starter cultures or applied as natural additives to improve the quality of various food products. In the present study, 43 strains were isolated from different plant-based fermented foods and identified by molecular methods. The species found were distinctively specific according to the food source. Only six Lactiplantibacillus plantarum strains, all isolated from sauerkraut, showed the ability to produce exopolysaccharide (EPS). The utilization of glucose, fructose and sucrose was explored with regard to EPS and biomass accumulation by the tested strains. Sucrose was clearly the best carbon source for EPS production by most of the strains, yielding up to 211.53 mg/L by strain Lactiplantibacillus plantarum ZE2, while biomass accumulation reached the highest levels in the glucose-based culture medium. Most strains produced similar levels of EPS with glucose and fructose, while fructose was utilized more poorly for biomass production, yielding about 50% of biomass compared to glucose for most strains. Composition analysis of the EPSs produced by strain Lactiplantibacillus plantarum ZE2 from glucose (EPS-1) and fructose (EPS-2) revealed that glucose (80-83 mol%) and protein (41% w/w) predominated in both analyzed EPSs. However, the yield of EPS-1 was twice higher than that of EPS-2, and differences in the levels of all detected sugars were found, which shows that even for the same strain, EPS yield and composition vary depending on the carbon source. These results may be the basis for the development of tailored EPS-producing starter cultures for food fermentations, as well as technologies for the production of EPS for various applications.