Quantitative isotope-dilution high-resolution-mass-apectrometry analysis of multiple intracellular metabolites in Clostridium autoethanogenum with uniformly 13C-labeled standards derived from Spirulina

We have investigated the applicability of commercially available lyophilized spirulina (Arthrospira platensis), a microorganism uniformly labeled with 13C, as a readily accessible source of multiple 13C-labeled metabolites suitable as internal standards for the quantitative determination of intracellular bacterial metabolites. Metabolites of interest were analyzed by hydrophilic-interaction liquid chromatography coupled with high-resolution mass spectrometry. Multiple internal standards obtained from uniformly (U)-13C-labeled extracts from spirulina were used to enable isotope-dilution mass spectrometry (IDMS) in the identification and quantification of intracellular metabolites. Extraction of the intracellular metabolites of Clostridium autoethanogenum using 2:1:1 chloroform/methanol/water was found to be the optimal method in comparison with freeze–thaw, homogenization, and sonication methods. The limits of quantification were ≤1 μM with excellent linearity for all of the calibration curves (R2 ≥ 0.99) for 74 metabolites. The precision and accuracy were found to be within relative standard deviations (RSDs) of 15% for 49 of the metabolites and within RSDs of 20% for all of the metabolites. The method was applied to study the effects of feeding different levels of carbon monoxide (as a carbon source) on the central metabolism and Wood–Ljungdahl pathway of C. autoethanogenum grown in continuous culture over 35 days. Using LC-IDMS with U-13C spirulina allowed the successful quantification of 52 metabolites in the samples, including amino acids, carboxylic acids, sugar phosphates, purines, and pyrimidines. The method provided absolute quantitative data on intracellular metabolites that was suitable for computational modeling to understand and optimize the C. autoethanogenum metabolic pathways active in gas fermentation

Moorella stamsii sp. nov., a new anaerobic thermophilic hydrogenogenic carboxydotroph isolated from digester sludge

A novel anaerobic, thermophilic, carbon monoxide-utilizing bacterium, strain E3-O, was isolated from anaerobic sludge of a municipal solid waste digester. Cells were straight rods, 0.6 to 1μm in diameter and 2 to 3 μm in length, growing as single cells or in pairs. Cells formed round terminal endospores. The temperature range for growth was 50 to 70°C, with an optimum at 65°C. The pH range for growth was 5.7 to 8.0, with an optimum at 7.5. Strain E3-O had the capability to ferment various sugars, such as fructose, galactose, glucose, mannose, raffinose, ribose, sucrose and xylose, producing mainly H2 and acetate. In addition, the isolate was able to grow with CO as the sole carbon and energy source. CO oxidation was coupled to H2 and CO2 formation. The G+C content of the genomic DNA was 54.6 mol %. Based on 16S rRNA gene sequence analysis, this bacterium is most closely related to Moorella glycerini (97% sequence identity). Based on the physiological features and phylogenetic analysis, it is proposed that strain E3-O should be classified in the genus Moorella as a new species, Moorella stamsii. The type strain of Moorella stamsii is E3-OT (=DMS 26271T=CGMCC 1.5181T).This work was possible through the financial support provided by the Portuguese Science Foundation (FCT) and the European Social Fund (POPH-QREN) through a PhD grant SFRH/BD/48965/2008 to J.I.A

Quantitative Bioreactor Monitoring of Intracellular Bacterial Metabolites in Clostridium autoethanogenum Using Liquid Chromatography–Isotope Dilution Mass Spectrometry

We report a liquid chromatography–isotope dilution mass spectrometry method for the simultaneous quantification of 131 intracellular bacterial metabolites of Clostridium autoethanogenum. A comprehensive mixture of uniformly 13C-labeled internal standards (U-13C IS) was biosynthesized from the closely related bacterium Clostridium pasteurianum using 4% 13C–glucose as a carbon source. The U-13C IS mixture combined with 12C authentic standards was used to validate the linearity, precision, accuracy, repeatability, limits of detection, and quantification for each metabolite. A robust-fitting algorithm was employed to reduce the weight of the outliers on the quantification data. The metabolite calibration curves were linear with R2 ≥ 0.99, limits of detection were ≤1.0 μM, limits of quantification were ≤10 μM, and precision/accuracy was within RSDs of 15% for all metabolites. The method was subsequently applied for the daily monitoring of the intracellular metabolites of C. autoethanogenum during a CO gas fermentation over 40 days as part of a study to optimize biofuel production. The concentrations of the metabolites were estimated at steady states of different pH levels using the robust-fitting mathematical approach, and we demonstrate improved accuracy of results compared to conventional regression. Metabolic pathway analysis showed that reactions of the incomplete (branched) tricarboxylic acid “cycle” were the most affected pathways associated with the pH shift in the bioreactor fermentation of C. autoethanogenum and the concomitant changes in ethanol production

Erratum:The behavioral and psychological symptoms of dementia in down syndrome scale (BPSD-DS II): Optimization and further validation

BACKGROUND: People with Down syndrome (DS) are at high risk to develop Alzheimer's disease dementia (AD). Behavioral and psychological symptoms of dementia (BPSD) are common and may also serve as early signals for dementia. However, comprehensive evaluation scales for BPSD, adapted to DS, are lacking. Therefore, we previously developed the BPSD-DS scale to identify behavioral changes between the last six months and pre-existing life-long characteristic behavior. OBJECTIVE: To optimize and further study the scale (discriminative ability and reliability) in a large representative DS study population. METHODS: Optimization was based on item irrelevance and clinical experiences obtained in the initial study. Using the shortened and refined BPSD-DS II, informant interviews were conducted to evaluate 524 DS individuals, grouped according to dementia status: no dementia (DS, N = 292), questionable dementia (DS + Q, N = 119), and clinically diagnosed dementia (DS + AD, N = 113). RESULTS: Comparing item change scores between groups revealed prominent changes in frequency and severity for anxious, sleep-related, irritable, restless/stereotypic, apathetic, depressive, and eating/drinking behavior. For most items, the proportion of individuals displaying an increased frequency was highest in DS + AD, intermediate in DS + Q, and lowest in DS. For various items within sections about anxious, sleep-related, irritable, apathetic, and depressive behaviors, the proportion of individuals showing an increased frequency was already substantial in DS + Q, suggesting that these changes may serve as early signals of AD in DS. Reliability data were promising. CONCLUSION: The optimized scale yields largely similar results as obtained with the initial version. Systematically evaluating BPSD in DS may increase understanding of changes among caregivers and (timely) adaptation of care/treatment

Isolation and characterization of a new CO-utilizing strain, Thermoanaerobacter thermohydrosulfuricus subsp. carboxydovorans, isolated from a geothermal spring in Turkey

A novel anaerobic, thermophilic, Gram-positive, spore-forming, and sugar-fermenting bacterium (strain TLO) was isolated from a geothermal spring in Ayaş, Turkey. The cells were straight to curved rods, 0.4–0.6 μm in diameter and 3.5–10 μm in length. Spores were terminal and round. The temperature range for growth was 40–80°C, with an optimum at 70°C. The pH optimum was between 6.3 and 6.8. Strain TLO has the capability to ferment a wide variety of mono-, di-, and polysaccharides and proteinaceous substrates, producing mainly lactate, next to acetate, ethanol, alanine, H2, and CO2. Remarkably, the bacterium was able to grow in an atmosphere of up to 25% of CO as sole electron donor. CO oxidation was coupled to H2 and CO2 formation. The G + C content of the genomic DNA was 35.1 mol%. Based on 16S rRNA gene sequence analysis and the DNA–DNA hybridization data, this bacterium is most closely related to Thermoanaerobacter thermohydrosulfuricus and Thermoanaerobacter siderophilus (99% similarity for both). However, strain TLO differs from Thermoanaerobacter thermohydrosulfuricus in important aspects, such as CO-utilization and lipid composition. These differences led us to propose that strain TLO represents a subspecies of Thermoanaerobacter thermohydrosulfuricus, and we therefore name it Thermoanaerobacter thermohydrosulfuricus subsp. carboxydovorans

Sequencing and Comparative Genome Analysis of Two Pathogenic Streptococcus gallolyticus Subspecies: Genome Plasticity, Adaptation and Virulence

Streptococcus gallolyticus infections in humans are often associated with bacteremia, infective endocarditis and colon cancers. The disease manifestations are different depending on the subspecies of S. gallolyticus causing the infection. Here, we present the complete genomes of S. gallolyticus ATCC 43143 (biotype I) and S. pasteurianus ATCC 43144 (biotype II.2). The genomic differences between the two biotypes were characterized with comparative genomic analyses. The chromosome of ATCC 43143 and ATCC 43144 are 2,36 and 2,10 Mb in length and encode 2246 and 1869 CDS respectively. The organization and genomic contents of both genomes were most similar to the recently published S. gallolyticus UCN34, where 2073 (92%) and 1607 (86%) of the ATCC 43143 and ATCC 43144 CDS were conserved in UCN34 respectively. There are around 600 CDS conserved in all Streptococcus genomes, indicating the Streptococcus genus has a small core-genome (constitute around 30% of total CDS) and substantial evolutionary plasticity. We identified eight and five regions of genome plasticity in ATCC 43143 and ATCC 43144 respectively. Within these regions, several proteins were recognized to contribute to the fitness and virulence of each of the two subspecies. We have also predicted putative cell-surface associated proteins that could play a role in adherence to host tissues, leading to persistent infections causing sub-acute and chronic diseases in humans. This study showed evidence that the S. gallolyticus still possesses genes making it suitable in a rumen environment, whereas the ability for S. pasteurianus to live in rumen is reduced. The genome heterogeneity and genetic diversity among the two biotypes, especially membrane and lipoproteins, most likely contribute to the differences in the pathogenesis of the two S. gallolyticus biotypes and the type of disease an infected patient eventually develops