Mark Lefsrud

Shotgun metaproteomics of the human distal gut microbiota

The human gut contains a dense, complex and diverse microbial community, comprising the gut microbiome. Metagenomics has recently revealed the composition of genes in the gut microbiome, but provides no direct information about which genes are expressed or functioning. Therefore, our goal was to develop a novel approach to directly identify microbial proteins in fecal samples to gain information about the genes expressed and about key microbial functions in the human gut. We used a non-targeted, shotgun mass spectrometry-based whole community proteomics, or metaproteomics, approach for the first deep proteome measurements of thousands of proteins in human fecal samples, thus demonstrating this approach on the most complex sample type to date. The resulting metaproteomes had a skewed distribution relative to the metagenome, with more proteins for translation, energy production and carbohydrate metabolism when compared to what was earlier predicted from metagenomics. Human proteins, including antimicrobial peptides, were also identified, providing a non-targeted glimpse of the host response to the microbiota. Several unknown proteins represented previously undescribed microbial pathways or host immune responses, revealing a novel complex interplay between the human host and its associated microbes.

Community Genomic and Proteomic Analyses of Chemoautotrophic Iron-Oxidizing “Leptospirillum rubarum” (Group II) and “Leptospirillum ferrodiazotrophum” (Group III) Bacteria in Acid Mine Drainage Biofilms

ABSTRACT We analyzed near-complete population (composite) genomic sequences for coexisting acidophilic iron-oxidizing Leptospirillum group II and III bacteria (phylum Nitrospirae) and an extrachromosomal plasmid from a Richmond Mine, Iron Mountain, CA, acid mine drainage biofilm. Community proteomic analysis of the genomically characterized sample and two other biofilms identified 64.6% and 44.9% of the predicted proteins of Leptospirillum groups II and III, respectively, and 20% of the predicted plasmid proteins. The bacteria share 92% 16S rRNA gene sequence identity and >60% of their genes, including integrated plasmid-like regions. The extrachromosomal plasmid carries conjugation genes with detectable sequence similarity to genes in the integrated conjugative plasmid, but only those on the extrachromosomal element were identified by proteomics. Both bacterial groups have genes for community-essential functions, including carbon fixation and biosynthesis of vitamins, fatty acids, and biopolymers (including cellulose); proteomic analyses reveal these activities. Both Leptospirillum types have multiple pathways for osmotic protection. Although both are motile, signal transduction and methyl-accepting chemotaxis proteins are more abundant in Leptospirillum group III, consistent with its distribution in gradients within biofilms. Interestingly, Leptospirillum group II uses a methyl-dependent and Leptospirillum group III a methyl-independent response pathway. Although only Leptospirillum group III can fix nitrogen, these proteins were not identified by proteomics. The abundances of core proteins are similar in all communities, but the abundance levels of unique and shared proteins of unknown function vary. Some proteins unique to one organism were highly expressed and may be key to the functional and ecological differentiation of Leptospirillum groups II and III.

Community proteogenomics highlights microbial strain-variant protein expression within activated sludge performing enhanced biological phosphorus removal

Enhanced biological phosphorus removal (EBPR) selects for polyphosphate accumulating microorganisms to achieve phosphate removal from wastewater. We used high-resolution community proteomics to identify key metabolic pathways in ‘Candidatus Accumulibacter phosphatis’ (A. phosphatis)-mediated EBPR and to evaluate the contributions of co-existing strains within the dominant population. Overall, 702 proteins from the A. phosphatis population were identified. Results highlight the importance of denitrification, fatty acid cycling and the glyoxylate bypass in EBPR. Strong similarity in protein profiles under anaerobic and aerobic conditions was uncovered (only 3% of A. phosphatis-associated proteins exhibited statistically significant abundance differences). By comprehensive genome-wide alignment of 13 930 orthologous proteins, we uncovered substantial differences in protein abundance for enzyme variants involved in both core-metabolism and EBPR-specific pathways among the A. phosphatis population. These findings suggest an essential role for genetic diversity in maintaining the stable performance of EBPR systems and, hence, demonstrate the power of integrated cultivation-independent genomics and proteomics for the analysis of complex biotechnological systems.

Variability in Elemental Accumulations Among Leafy Brassica oleracea Cultivars and Selections

Abstract The vegetable brassicas are consumed in part for their nutritional values of calcium (Ca), magnesium (Mg), potassium (K), iron (Fe), and zinc (Zn). However, information on the genetic variability of elemental accumulation within kale and collards (Brassica oleracea L. var. acephala DC) is limited. Therefore, 22 kale and collard cultivars and selections suitable for the northeastern United States were field grown under similar fertility regimes over two years and evaluated for elemental accumulation. Leaf tissues were analyzed using inductively coupled argon plasma–atomic emission spectrometry. Significant variability among cultivars and selections was observed for tissue Ca, Mg, K, Fe, and Zn. On average, a two-fold difference in elemental accumulation among the cultivars and selections was measured. Tissue Ca levels ranged from 1.2 (“Crimson Garden”) to 3.1% (“NZ Thousand Head”), tissue Mg ranged from 0.3 (“Crimson Garden”) to 0.6% (“NZ Thousand Head”), tissue K ranged from 2.1 (“NZ Thousand Head”) to 3.5% (30665-96G11), tissue Fe ranged from 53.1 (“Winterbor F1”) to 114.2 mg/kg (“Giant Jersey Kale”), and tissue Zn ranged from 29.1 (“Shetland”) to 71.9 mg/kg (“Redbor F1”). Significant year-to-year variability occurred for Ca, Mg, Fe, and Zn accumulation. Despite these yearly changes, ranking of the cultivars and selections for elemental accumulation, as determined by Spearmans rank correlation coefficient, did not change from year 1 to year 2. Overall, the cultivar with the highest elemental leaf accumulation was “Redbor F1”. Information on genotypic variability for elemental accumulation may be important for producers and consumers looking to select kale and collards with higher nutritional levels of beneficial dietary elements.

Nitrogen Levels Influence Biomass, Elemental Accumulations, and Pigment Concentrations in Spinach

ABSTRACT Spinach (Spinacia oleracea L.) has one of the highest United States per capita consumption rates among leafy vegetable crops, and also ranks second for lutein and β-carotene carotenoid concentration. The objectives of this study were to determine the effects of nitrogen (N) concentration on elemental and pigment accumulation in spinach. Two spinach cultivars (‘Melody’ and ‘Springer F1’) were greenhouse grown in nutrient solution culture under N treatments of 13, 26, 52, and 105 mg L− 1. Leaf tissue biomass increased from 45.6 to 273.2 g plant− 1 and from 127.0 to 438.6 g plant− 1 as N increased from 13 to 105 mg L− 1 for ‘Springer F1’ and ‘Melody’, respectively. Leaf tissue N, phosphorus (P), calcium (Ca), magnesium (Mg), copper (Cu), and zinc (Zn) responded to N treatments. Lutein accumulations, expressed on a fresh weight basis, responded quadratically to increasing N treatments for ‘Springer F1’. Maximum lutein values were 110 and 76 μ g g− 1 on a fresh weight basis, and maximum β-carotene values were 85 and 57 μ g g− 1 on a fresh weight basis for ‘Springer F1’ and ‘Melody’, respectively. Interestingly, N levels had a significant effect on carotenoid accumulation in both ‘Springer F1’ and ‘Melody’ when the pigments were expressed on a dry weight basis. Leaf tissue lutein increased from 0.59 to 1.06 mg g− 1 and from 0.59 to 0.90 mg g− 1 on a dry weight basis with increasing N treatments for ‘Springer F1’ and ‘Melody’, respectively. Reporting lutein and β-carotene on both a fresh and dry weight basis may be the most accurate way to express the carotenoid values of spinach.

Comparative shotgun proteomic analysis of Clostridium acetobutylicum from butanol fermentation using glucose and xylose

BackgroundButanol is a second generation biofuel produced by Clostridium acetobutylicum through acetone-butanol-ethanol (ABE) fermentation process. Shotgun proteomics provides a direct approach to study the whole proteome of an organism in depth. This paper focuses on shotgun proteomic profiling of C. acetobutylicum from ABE fermentation using glucose and xylose to understand the functional mechanisms of C. acetobutylicum proteins involved in butanol production.ResultsWe identified 894 different proteins in C. acetobutylicum from ABE fermentation process by two dimensional - liquid chromatography - tandem mass spectrometry (2D-LC-MS/MS) method. This includes 717 proteins from glucose and 826 proteins from the xylose substrate. A total of 649 proteins were found to be common and 22 significantly differentially expressed proteins were identified between glucose and xylose substrates.ConclusionOur results demonstrate that flagellar proteins are highly up-regulated with glucose compared to xylose substrate during ABE fermentation. Chemotactic activity was also found to be lost with the xylose substrate due to the absence of CheW and CheV proteins. This is the first report on the shotgun proteomic analysis of C. acetobutylicum ATCC 824 in ABE fermentation between glucose and xylose substrate from a single time data point and the number of proteins identified here is more than any other study performed on this organism up to this report.

Microalgae for phosphorus removal and biomass production: a six species screen for dual‐purpose organisms

Microalgae biofuel production can be feasible when a second function is added, such as wastewater treatment. Microalgae differ in uptake of phosphorus (P) and growth, making top performer identification fundamental. The objective of this screen was to identify dual‐purpose candidates capable of high rates of P removal and growth. Three freshwater – Chlorella sp., Monoraphidium minutum sp., and Scenedesmus sp. – and three marine – Nannochloropsis sp., N. limnetica sp., and Tetraselmis suecica sp. – species were batch cultured in 250 mL flasks over 16 days to quantitate total phosphorus (TP) removal and growth as a function of P loads (control, and 5, 10, and 15 mg L−1 enrichment of control). Experimental design used 100 μmol m−2 s−1 of light, a light/dark cycle of 14/10 h, and no CO2 enrichment. Phosphorus uptake was dependent on species, duration of exposure, and treatment, with significant interaction effects. Growth was dependant on species and treatment. Not all species showed increased P removal with increasing P addition, and no species demonstrated higher growth. Nannochloropsis sp and N. limnetica sp. performed poorly across all treatments. Two dual‐purpose candidates were identified. At the 10 mg L−1 treatment Monoraphidium minutum sp. removed 67.1% (6.66 mg L−1 ± 0.60 SE) of TP at day 8, 79.3% (7.86 mg L−1 ± 0.28 SE) at day 16, and biomass accumulation of 0.63 g L−1 ± 0.06 SE at day 16. At the same treatment Tetraselmis suecica sp. removed 79.4% (6.98 mg L−1 ± 0.24 SE) TP at day 8, 83.0% (7.30 mg L−1 ± 0.60 SE) at day 16, and biomass of 0.55 g L−1 ± 0.02 SE at day 16. These species merit further study using high‐density wastewater cultures and lipid profiling to assess suitability for a nutrient removal and biomass/biofuel production scheme.

Fatty acid profiling of the seed oils of some varieties of field peas (Pisum sativum) by RP-LC/ESI-MS/MS: towards the development of an oilseed pea.

Reversed-phase liquid chromatography coupled to negative-ion electrospray tandem mass spectrometry (RP-LC/ESI-MS/MS) was used to study the fatty acid profile from the oil of harvested field pea (Pisum sativum) varieties as part of a research project to develop this legume as a commercial oilseed for Canada. The seed oils from pea samples contained palmitic and stearic acids as major saturated fatty acids. Oleic, linoleic and linolenic acids were the major unsaturated fatty acids found. Small percentages of other long chain fatty acids were also detected. This profile suggests that the species of field pea investigated might have the potential to be used as raw materials to develop a future new oilseed crop for the food industry. Fatty acid extracts did not require further manipulation before final analysis by RP-LC/ESI-MS/MS, indicating the utility and relative simplicity of this technique for future screening studies.

Guidelines for measuring and reporting environmental parameters for experiments in greenhouses

BackgroundThe importance of appropriate, accurate measurement and reporting of environmental parameters in plant sciences is a significant aspect of quality assurance for all researchers and their research. There is a clear need for ensuring research across the world can be compared, understood and where necessary replicated by fellow researchers. A common set of guidelines to educate, assist and encourage comparativeness is of great importance. On the other hand, the level of effort and attention to detail by an individual researcher should be commensurate with the particular research being conducted. For example, a researcher focusing on interactions of light and temperature should measure all relevant parameters and report a measurement summary that includes sufficient detail allowing for replication. Such detail may be less relevant when the impact of environmental parameters on plant growth and development is not the main research focus. However, it should be noted that the environmental experience of a plant during production can have significant impact when subsequent experiments investigate plants at a molecular, biochemical or genetic level or where species interactions are considered. Thus, researchers are encouraged to make a critical assessment of what parameters are of primary importance in their research and these parameters should be measured and reported.ContentThis paper brings together a collection of parameters that the authors, as members of International Committee on Controlled Environment Guidelines (ICCEG) in consultation with members of our three parent organizations, believe constitute those which should be recorded and reported when publishing scientific data from experiments in greenhouses. It provides recommendations to end users on when, how and where these parameters should be measured along with the appropriate internationally standardized units that should be used.

Shotgun proteomic monitoring of Clostridium acetobutylicum during stationary phase of butanol fermentation using xylose and comparison with the exponential phase

Economically viable production of solvents through acetone–butanol–ethanol (ABE) fermentation requires a detailed understanding of Clostridium acetobutylicum. This study focuses on the proteomic profiling of C. acetobutylicum ATCC 824 from the stationary phase of ABE fermentation using xylose and compares with the exponential growth by shotgun proteomics approach. Comparative proteomic analysis revealed 22.9% of the C. acetobutylicum genome and 18.6% was found to be common in both exponential and stationary phases. The proteomic profile of C. acetobutylicum changed during the ABE fermentation such that 17 proteins were significantly differentially expressed between the two phases. Specifically, the expression of five proteins namely, CAC2873, CAP0164, CAP0165, CAC3298, and CAC1742 involved in the solvent production pathway were found to be significantly lower in the stationary phase compared to the exponential growth. Similarly, the expression of fucose isomerase (CAC2610), xylulose kinase (CAC2612), and a putative uncharacterized protein (CAC2611) involved in the xylose utilization pathway were also significantly lower in the stationary phase. These findings provide an insight into the metabolic behavior of C. acetobutylicum between different phases of ABE fermentation using xylose.