Time filter

Source Type

National City, IL, United States

Naumann T.A.,National United University | Naldrett M.J.,Donald Danforth Plant Science Center | Ward T.J.,National United University | Price N.P.J.,Renewable Product Technology Research Unit
Protein Science | Year: 2015

Polyglycine hydrolases are secreted fungal proteases that cleave glycine-glycine peptide bonds in the inter-domain linker region of specific plant defense chitinases. Previously, we reported the catalytic activity of polyglycine hydrolases from the phytopathogens Epicoccum sorghi (Es-cmp) and Cochliobolus carbonum (Bz-cmp). Here we report the identity of their encoding genes and the primary amino acid sequences of the proteins responsible for these activities. Peptides from a tryptic digest of Es-cmp were analyzed by LC-MS/MS and the spectra obtained were matched to a draft genome sequence of E. sorghi. From this analysis, a 642 amino acid protein containing a predicted β-lactamase catalytic region of 280 amino acids was identified. Heterologous strains of the yeast Pichia pastoris were created to express this protein and its homolog from C. carbonum from their cDNAs. Both strains produced recombinant proteins with polyglycine hydrolase activity as shown by SDS-PAGE and MALDI-MS based assays. Site directed mutagenesis was used to mutate the predicted catalytic serine of Es-cmp to glycine, resulting in loss of catalytic activity. BLAST searching of publicly available fungal genomes identified full-length homologous proteins in 11 other fungi of the class Dothideomycetes, and in three fungi of the related class Sordariomycetes while significant BLAST hits extended into the phylum Basidiomycota. Multiple sequence alignment led to the identification of a network of seven conserved tryptophans that surround the β-lactamase-like region. This is the first report of a predicted β-lactamase that is an endoprotease. © 2015 The Protein Society. Source

Edrington T.S.,Food and Feed Safety Research Unit | Bischoff K.M.,Renewable Product Technology Research Unit | Loneragan G.H.,Texas Tech University | Nisbet D.J.,Food and Feed Safety Research Unit
Journal of Animal Science | Year: 2014

Dried distiller's grains (DG) produced from ethanol fermentations dosed with 0 (control), 2, or 20 mg/kg virginiamycin-based product or spiked with virginiamycin (VM) postfermentation were fed to cattle and effects on antimicrobial susceptibility, and prevalence of antimicrobial resistance genes in commensal bacteria was examined. Biological activity assays of DG (from each fermentation) indicated a concentration of 0, 0.7, and 8.9 mg/kg VM, respectively. Twenty-four crossbred beef steers were fed 1 of 4 diets (containing 8% of each of the different batches of DG) and a fourth using 8% of the control DG (0 mg/kg VM) + 0.025 g/kg V-Max50 (positive control) for 7 wk. Fecal samples were collected weekly throughout the experimental period and cultured for Escherichia coli and Enterococcus, and isolates were examined for antimicrobial susceptibility, antimicrobial resistance genes (vatE, ermB, and msrC in Enterococcus), and integrons (E. coli). No treatment differences (P > 0.05) were observed in antimicrobial susceptibility of the E. coli isolates. Enterococcus isolates were resistant to more antimicrobials; however, this was influenced by the species of Enterococcus and not treatment (P > 0.10). The prevalence of ermB was greater (P < 0.05) in the control isolates after 4 and 6 wk while at wk 7, prevalence was greater (P < 0.01) in the 0.7 and 8.9 mg/kg VM treatments. Taken together, the minor treatment differences observed for the presence of ermB coupled with the lack of effect on antimicrobial susceptibility patterns suggest that feeding DG containing VM residues should have minimal if any impact on prevalence of antimicrobial resistance. © 2014 American Society of Animal Science. All rights reserved. Source

Naumann T.A.,Bacterial Foodborne Pathogens and Mycology Research Unit | Wicklow D.T.,Bacterial Foodborne Pathogens and Mycology Research Unit | Price N.P.,Renewable Product Technology Research Unit
Biochemical Journal | Year: 2014

Cmps (chitinase-modifying proteins) are fungal proteases that truncate plant class IV chitinases by cleaving near their Ntermini. We previously described Fv-cmp, a fungalysin protease that cleaves a conserved glycine-cysteine bond within the hevein domain. In the present paper we describe a new type of cmp, polyglycine hydrolases, as proteases that selectively cleave glycine-glycine peptide bonds within the polyglycine linker of plant class IV chitinases. Polyglycine hydrolases were purified from Cochliobolus carbonum (syn. Bipolaris zeicola; Bz-cmp) and Epicoccum sorghi (syn. Phoma sorghina; Es-cmp) and were shown to cleave three different maize class IV chitinase substrates. The proteolytic cleavage sites were assessed by SDS/PAGE and MALDI-TOF-MS and indicated the cleavage of multiple peptide bonds within the polyglycine linker regions. Site-directed mutagenesis was used to produce mutants of maize ChitB chitinase in which two serine residues in its linker were systematically modified to glycine. Serine to glycine changes in the ChitB linker resulted in higher susceptibility to truncation by Bz-cmp and altered substrate specificity for Bz-cmp and Es-cmp, such that different glycine-glycine peptide bonds were cleaved. Removal of the hevein domain led to loss of Es-cmp activity, indicating that interactions outside of the active site are important for recognition. Our findings demonstrate that plant class IV chitinases with polyglycine linkers are targeted for truncation by selective polyglycine hydrolases that are secreted by plant pathogenic fungi. This novel proteolysis of polyglycine motifs is previously unreported, but the specificity is similar to that of bacterial lysostaphin proteases, which cleave pentaglycine crosslinks from peptidoglycan. © 2014 Biochemical Society. Source

Appell M.,National United University | Jackson M.A.,Renewable Product Technology Research Unit | Wang L.C.,National United University | Ho C.-H.,Central Michigan University | Mueller A.,Central Michigan University
Journal of Separation Science | Year: 2014

A new LC method to detect fusaric acid (FA) in maize is reported based on a molecularly imprinted SPE clean-up using mimic-templated molecularly imprinted polymers. Picolinic acid was used as a toxin analog for imprinting polymers during a thermolytic synthesis. Both acidic and basic functional monomers were predicted to have favorable binding interactions by MP2 ab initio calculations. Imprinted polymers synthesized with methacrylic acid or 2-dimethylaminoethyl methacrylate exhibited imprinting effects in SPE analysis. FA levels were determined using RP ion-pairing chromatography with diode-array UV detection and tetrabutylammonium hydrogen sulfate in the mobile phase. A method was developed to detect FA in maize using molecularly imprinted SPE analysis within the range of 1-100 μg/g with recoveries between 83.9 and 92.1%. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Naumann T.A.,Bacterial Foodborne Pathogens and Mycology Research Unit | Price N.P.J.,Renewable Product Technology Research Unit
Molecular Plant Pathology | Year: 2012

Plant class IV chitinases have a small amino-terminal chitin-binding domain and a larger chitinase domain, and are involved in plant defence against fungal infection. Our previous work on the chitinases ChitA and ChitB from the model monocotyledon Zea mays showed that the chitin-binding domain is removed by secreted fungal proteases called fungalysins. In this article, we extend this work to dicotyledons. The effects of fungalysin-like proteases on four class IV chitinases from the model dicotyledon Arabidopsis thaliana were analysed. Four Arabidopsis chitinases were heterologously expressed in Pichia pastoris, purified and shown to have chitinase activity against a chitohexaose (dp6) substrate. The incubation of these four chitinases with Fv-cmp, a fungalysin protease secreted by Fusarium verticillioides, resulted in the truncation of AtchitIV3 and AtchitIV5. Moreover, incubation with secreted proteins from Alternaria brassicae, a pathogen of A.thaliana and brassica crops, also led to a similar truncation of AtchitIV3 and AtchitIV4. Our finding that class IV chitinases from both dicotyledons (A.thaliana) and monocotyledons (Z.mays) are truncated by proteases secreted by specialized pathogens of each plant suggests that this may be a general mechanism of plant-fungal pathogenicity. © 2012 BSPP AND BLACKWELL PUBLISHING LTD. Source

Discover hidden collaborations