L. Brent Selinger

Differential expression of proteins in response to the interaction between the pathogen Fusarium graminearum and its host, Hordeum vulgare

Using proteomic techniques, a study aimed at isolating and identifying proteins associated with resistance to fusarium head blight (FHB) was conducted on six barley genotypes of varying resistance. At anthesis, barley spikelets were point inoculated with Fusarium graminearum macroconidial suspensions or mock inoculum. In total, 43 acidic protein spots out of 600 were detected 3 days postinoculation to be differentially expressed due to FHB and were identified. Identification of proteins responsive to FHB included those associated with oxidative burst and oxidative stress response, such as malate dehydrogenase and peroxidases, and pathogenesis‐related (PR). An increase in abundance of PR‐3 or PR‐5 could be associated with the resistant genotypes CI4196, Svansota, and Harbin, as well as the intermediate resistant genotype CDC Bold. On the contrary, the susceptible genotype Stander showed a decrease in abundance of these acidic PR‐proteins. In the susceptible and intermediate resistant genotypes Stander and CDC Bold, as well as CI4196, the increased abundance of proteins associated with an oxidative response might have prepared the terrain for saprophytic fungal invasion. On the contrary, in the resistant sources Harbin and Svansota we did not observed change in abundance of these proteins. Not a single significant change in acidic protein abundance could be detected in Chevron. Three distinct response patterns are reported from these six barley genotypes.

Snapshot of the Eukaryotic Gene Expression in Muskoxen Rumen—A Metatranscriptomic Approach

Background Herbivores rely on digestive tract lignocellulolytic microorganisms, including bacteria, fungi and protozoa, to derive energy and carbon from plant cell wall polysaccharides. Culture independent metagenomic studies have been used to reveal the genetic content of the bacterial species within gut microbiomes. However, the nature of the genes encoded by eukaryotic protozoa and fungi within these environments has not been explored using metagenomic or metatranscriptomic approaches. Methodology/Principal Findings In this study, a metatranscriptomic approach was used to investigate the functional diversity of the eukaryotic microorganisms within the rumen of muskoxen (Ovibos moschatus), with a focus on plant cell wall degrading enzymes. Polyadenylated RNA (mRNA) was sequenced on the Illumina Genome Analyzer II system and 2.8 gigabases of sequences were obtained and 59129 contigs assembled. Plant cell wall degrading enzyme modules including glycoside hydrolases, carbohydrate esterases and polysaccharide lyases were identified from over 2500 contigs. These included a number of glycoside hydrolase family 6 (GH6), GH48 and swollenin modules, which have rarely been described in previous gut metagenomic studies. Conclusions/Significance The muskoxen rumen metatranscriptome demonstrates a much higher percentage of cellulase enzyme discovery and an 8.7x higher rate of total carbohydrate active enzyme discovery per gigabase of sequence than previous rumen metagenomes. This study provides a snapshot of eukaryotic gene expression in the muskoxen rumen, and identifies a number of candidate genes coding for potentially valuable lignocellulolytic enzymes.

Diets Enriched in Oat Bran or Wheat Bran Temporally and Differentially Alter the Composition of the Fecal Community of Rats

A clear understanding of how diet alters gastrointestinal communities is important given the suggested link between gut community composition and a wide variety of disease pathologies. To characterize this link for commonly consumed dietary fiber sources, we investigated the change in the fecal community of rats fed diets containing 5% nonnutritive fiber (control), 3% (wt:wt) oat bran plus 2% nonnutritive fiber (OB), or 5% (w/w) wheat bran (WB) over a 28-d feeding trial using both molecular- and cultivation-based methodologies. Pooled fecal samples from 8 rats fed the same diet were analyzed at 4 time points. On d 28, bran-fed rats had approximately twice the total cultivable bacteria than rats fed the control diet. Over the course of feeding, the cultivable community was initially dominated by bacteroides, then by bifidobacteria, lactobacilli, enterococci, and various enterics. In contrast, molecular analysis revealed the appearance of new operational taxonomic units (phylotypes) that were both temporally and inequitably distributed throughout the fecal community. The majority of change occurred in 2 major lineages within the Firmicutes: the Clostridium coccoides group and the Clostridium leptum subgroup. The time course of change depended on the source of bran, with the majority of new phylotypes appearing by d 14 (OB) or d 28 (WB), although adaptation of the fecal community was slow and continued over the entire feeding trial. Bacterial community richness was higher in bran-fed rats than in those fed the control diet. Change within the C. coccoides and C. leptum lineages likely reflect their high abundance within the gut bacterial community and the role of clostridia in fiber digestion. The results illustrate the limitations of relying solely on cultivation to assess bacterial changes and illustrate that community changes are complex in an ecosystem containing high numbers of interdependent and competing species of bacteria.

Escherichia coli O157:H7 Strain Origin, Lineage, and Shiga Toxin 2 Expression Affect Colonization of Cattle†

ABSTRACT Enterohemorrhagic Escherichia coli O157:H7 has evolved into an important human pathogen with cattle as the main reservoir. The recent discovery of E. coli O157:H7-induced pathologies in challenged cattle has suggested that previously discounted bacterial virulence factors may contribute to the colonization of cattle. The objective of the present study was to examine the impact of lineage type, cytotoxin activity, and cytotoxin expression on the amount of E. coli O157:H7 colonization of cattle tissue and cells in vitro. Using selected bovine- and human-origin strains, we determined that lineage type predicted the amount of E. coli O157:H7 strain colonization: lineage I > intermediate lineages > lineage II. All E. coli O157:H7 strain colonization was dose dependent, with threshold colonization at 103 to 105 CFU and maximum colonization at 107 CFU. We also determined that an as-yet-unknown factor of strain origin was the most dominant predictor of the amount of strain colonization in vitro. The amount of E. coli O157:H7 colonization was also influenced by strain cytotoxin activity and the inclusion of cytotoxins from lineage I or intermediate lineage strains increased colonization of a lineage II strain. There was a higher level of expression of the Shiga toxin 1 gene (stx1) in human-origin strains than in bovine-origin strains. In addition, lineage I strains expressed higher levels of the Shiga toxin 2 gene (stx2). The present study supports a role for strain origin, lineage type, cytotoxin activity, and stx2 expression in modulating the amount of E. coli O157:H7 colonization of cattle.

Influence of Loss‐on‐Ignition Temperature and Heating Time on Ash Content of Compost and Manure

Abstract Loss‐on‐ignition (LOI) is a simple method for determining ash content, and by reciprocation, organic matter content of compost and manure. However, reported ignition temperatures and heating times for LOI measurements vary widely, and this brings into question the accuracy of one specific combination of ignition temperature and heating time over another. This study examined the effect of 42 temperature‐heating time combinations (six ignition temperatures from 400 to 650°C in 50°C increments by seven heating times of 1, 2, 8, 12, 16, 20, and 24‐h) on the ash content of a finished compost and a fresh manure. The experiment included the 550°C for 2‐h method recommended in Test Methods for Evaluation of Compost and Composting. The magnitude of the decrease in ash content due to increase in temperature was not consistent across all heating times. For example, after a 1‐h heating time for compost, ash content was 75.7% at 400°C and 67.5% at 650°C, compared to 69.6% at 400°C and 66.8% at 650°C after 24‐h. Irrespective of heating time, an ignition temperature of 400°C overestimated ash content for both compost and manure compared to the TMECC method. The TMECC method with its moderate temperature and short heating time requirement could reduce energy costs without affecting ash content results.

Kinetic and structural analysis of a bacterial protein tyrosine phosphatase‐like myo‐inositol polyphosphatase

PhyA from Selenomonas ruminantium (PhyAsr), is a bacterial protein tyrosine phosphatase (PTP)‐like inositol polyphosphate phosphatase (IPPase) that is distantly related to known PTPs. PhyAsr has a second substrate binding site referred to as a standby site and the P‐loop (HCX5R) has been observed in both open (inactive) and closed (active) conformations. Site‐directed mutagenesis and kinetic and structural studies indicate PhyAsr follows a classical PTP mechanism of hydrolysis and has a broad specificity toward polyphosphorylated myo‐inositol substrates, including phosphoinositides. Kinetic and molecular docking experiments demonstrate PhyAsr preferentially cleaves the 3‐phosphate position of Ins P6 and will produce Ins(2)P via a highly ordered series of sequential dephosphorylations: D‐Ins(1,2,4,5,6)P5, Ins(2,4,5,6)P4, D‐Ins(2,4,5)P3, and D‐Ins(2,4)P2. The data support a distributive enzyme mechanism and suggest the PhyAsr standby site is involved in the recruitment of substrate. Structural studies at physiological pH and high salt concentrations demonstrate the “closed” or active P‐loop conformation can be induced in the absence of substrate. These results suggest PhyAsr should be reclassified as a D‐3 myo‐inositol hexakisphosphate phosphohydrolase and suggest the PhyAsr reaction mechanism is more similar to that of PTPs than previously suspected.

Diversity of Phytases in the Rumen

Examples of a new class of phytase related to protein tyrosine phosphatases (PTP) were recently isolated from several anaerobic bacteria from the rumen of cattle. In this study, the diversity of PTP-like phytase gene sequences in the rumen was surveyed by using the polymerase chain reaction (PCR). Two sets of degenerate primers were used to amplify sequences from rumen fluid total community DNA and genomic DNA from nine bacterial isolates. Four novel PTP-like phytase sequences were retrieved from rumen fluid, whereas all nine of the anaerobic bacterial isolates investigated in this work contained PTP-like phytase sequences. One isolate, Selenomonas lacticifex, contained two distinct PTP-like phytase sequences, suggesting that multiple phytate hydrolyzing enzymes are present in this bacterium. The degenerate primer and PCR conditions described here, as well as novel sequences obtained in this study, will provide a valuable resource for future studies on this new class of phytase. The observed diversity of microbial phytases in the rumen may account for the ability of ruminants to derive a significant proportion of their phosphorus requirements from phytate.

Non-therapeutic administration of a model antimicrobial growth promoter modulates intestinal immune responses

BackgroundThe development of efficacious alternatives to antimicrobial growth promoters (AGP) in livestock production is an urgent issue, but is hampered by a lack of knowledge regarding the mode of action of AGP. The belief that AGP modulate the intestinal microbiota has become prominent in the literature; however, there is a lack of experimental evidence to support this hypothesis. Using a chlortetracycline-murine-Citrobacter rodentium model, the ability of AGP to modulate the intestinal immune system in mammals was investigated.ResultsC. rodentium was transformed with the tetracycline resistance gene, tet O, and continuous oral administration of a non-therapeutic dose of chlortetracycline to mice did not affect densities of C. rodentium CFU in feces throughout the experiment or associated with mucosal surfaces in the colon (i.e. at peak and late infection). However, chlortetracycline regulated transcription levels of Th1 and Th17 inflammatory cytokines in a temporal manner in C. rodentium-inoculated mice, and ameliorated weight loss associated with infection. In mice inoculated with C. rodentium, those that received chlortetracycline had less pathologic changes in the distal colon than mice not administered CTC (i.e. relative to untreated mice). Furthermore, chlortetracycline administration at a non-therapeutic dose did not impart either prominent or consistent effects on the colonic microbiota.ConclusionData support the hypothesis that AGP function by modulating the intestinal immune system in mammals. This finding may facilitate the development of biorationale-based and efficacious alternatives to AGP.

A protein tyrosine phosphatase-like inositol polyphosphatase from Selenomonas ruminantium subsp. lactilytica has specificity for the 5-phosphate of myo-inositol hexakisphosphate.

Although it is becoming well known that myo-inositol polyphosphates and the enzymes involved in their metabolism play a critical role in eukaryotic systems, little is understood of their significance in prokaryotic systems. A novel protein tyrosine phosphatase (PTP)-like inositol polyphosphatase (IPPase) gene has been cloned from Selenomonas ruminantium subsp. lactilytica (phyAsrl). The deduced amino acid sequence of PhyAsrl is most similar to a PTP-like IPPase from the anaerobic bacterium S. ruminantium (35% identity), but also shows similarity (19-30% identity) to various other putative prokaryotic PTPs. Recombinant PhyAsrl could dephosphorylate myo-inositol hexakisphosphate (Ins P(6)) in vitro, and maximal activity was displayed at an ionic strength of 200 mM, a pH of 4.5, and a temperature of 55 degrees C. In order to elucidate its substrate specificity and pathway of Ins P(6) dephosphorylation, a combination of kinetic and high-performance ion-pair chromatography studies were conducted. The data indicated that PhyAsrl has a general specificity for polyphosphorylated myo-inositol substrates, but can also dephosphorylate molecules containing high energy pyrophosphate bonds in vitro. PhyAsrl is unique from other microbial IPPases in that it preferentially cleaves the 5-phosphate position of Ins P(6). Furthermore, it can produce Ins(2)P via a highly unique and ordered pathway of sequential dephosphorylation: Ins P(6), Ins(1,2,3,4,6)P(5), D-Ins(1,2,3,6)P(4), Ins(1,2,3)P(3), and D/L-Ins(1,2)P(2). Finally, reverse transcription PCR was used to determine that phyAsrl is constitutively expressed, and together with bioinformatic analysis, was used to gain an understanding of its physiological significance.

Escherichia coli O157:H7 super-shedder and non-shedder feedlot steers harbour distinct fecal bacterial communities.

Escherichia coli O157:H7 is a major foodborne human pathogen causing disease worldwide. Cattle are a major reservoir for this pathogen and those that shed E. coli O157:H7 at >104 CFU/g feces have been termed “super-shedders”. A rich microbial community inhabits the mammalian intestinal tract, but it is not known if the structure of this community differs between super-shedder cattle and their non-shedding pen mates. We hypothesized that the super-shedder state is a result of an intestinal dysbiosis of the microbial community and that a “normal” microbiota prevents E. coli O157:H7 from reaching super-shedding levels. To address this question, we applied 454 pyrosequencing of bacterial 16S rRNA genes to characterize fecal bacterial communities from 11 super-shedders and 11 contemporary pen mates negative for E. coli O157:H7. The dataset was analyzed by using five independent clustering methods to minimize potential biases and to increase confidence in the results. Our analyses collectively indicated significant variations in microbiome composition between super-shedding and non-shedding cattle. Super-shedders exhibited higher bacterial richness and diversity than non-shedders. Furthermore, seventy-two operational taxonomic units, mostly belonging to Firmicutes and Bacteroidetes phyla, were identified showing differential abundance between these two groups of cattle. The operational taxonomic unit affiliation provides new insight into bacterial populations that are present in feces arising from super-shedders of E. coli O157:H7.