Scott S. Crupper

5-Enolpyruvylshikimate-3-phosphate synthase from Staphylococcus aureus is insensitive to glyphosate.

The enzyme 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) catalyzes the penultimate step of the shikimate pathway, and is the target of the broad‐spectrum herbicide glyphosate. Kinetic analysis of the cloned EPSPS from Staphylococcus aureus revealed that this enzyme exerts a high tolerance to glyphosate, while maintaining a high affinity for its substrate phosphoenolpyruvate. Enzymatic activity is markedly influenced by monovalent cations such as potassium or ammonium, which is due to an increase in catalytic turnover. However, insensitivity to glyphosate appears to be independent from the presence of cations. Therefore, we propose that the Staphylococcus aureus EPSPS should be classified as a class II EPSPS. This research illustrates a critical mechanism of glyphosate resistance naturally occurring in certain pathogenic bacteria.

Escherichia coli Expressing EAST1 Toxin Did Not Cause an Increase of cAMP or cGMP Levels in Cells, and No Diarrhea in 5-Day Old Gnotobiotic Pigs

Background Enterotoxigenic Escherichia coli (ETEC) strains are the leading bacterial cause of diarrhea to humans and farm animals. These ETEC strains produce heat-labile toxin (LT) and/or heat-stable toxins that include type I (STa), type II (STb), and enteroaggregative heat-stable toxin 1 (EAST1). LT, STa, and STb (in pigs) are proven the virulence determinants in ETEC diarrhea. However, significance of EAST1 in ETEC-associated diarrheal has not been determined, even though EAST1 is highly prevalent among ETEC strains. Methodology/Principal Findings In this study, we constructed E. coli strains to express EAST1 toxin as the only toxin and studied them in cell lines and five-day old gnotobiotic piglets to determine significance of EAST1 toxin. Data from in vitro studies indicated that EAST1 did not stimulate an increase of intracellular cyclic AMP or GMP levels in T-84 cells or porcine cell line IPEC-J2, nor did it enhance LT or STa toxin of ETEC strains in stimulation of cAMP or cGMP in T-84 cells. In addition, 5-day old gnotobiotic pigs challenged with E. coli strains expressing EAST1 as the only toxin did not developed diarrhea or signs of clinical disease during 72 h post-inoculation. Conclusion/Significance Results from this study indicated that EAST1 alone is not sufficient to cause diarrhea in five-day old gnotobiotic pigs, and suggest that EAST1 likely is not a virulence determinant in ETEC-associated diarrhea.

Antimicrobial susceptibility of staphylococci isolated from the faeces of wild turkeys (Meleagris gallopavo)

Aims: The purpose of this study was to investigate the staphylococcal flora associated with wild turkey populations.

Testing a non-lethal DNA isolation technique for freshwater mussels that is compatible with polymerase chain reaction (PCR) based studies

Abstract In this study, a non-lethal DNA isolation technique was developed for freshwater mussels. A total of 45 Quadrula quadrula, 21 Q. metanevra, and 19 Q. pustulosa were collected from the Neosho and Verdigris Rivers in eastern Kansas. DNA was successfully isolated from 82 mussels using the non-lethal technique developed in this study. Spectrophotometer analysis of the 82 DNA samples resulted in an average DNA concentration of 488 ng/μl. Agarose gel electrophoresis of polymerase chain reaction products obtained using the purified mussel DNA demonstrated high quality, reproducible amplification products. All mussels remained alive for several months after tissue biopsy in the laboratory. A mark and recapture study was also performed to confirm survivability, thereby ensuring our procedure was non-lethal. Recapture efforts resulted in a 56% live recovery of mussels from the Neosho River and a 78% live recovery from mussels in the Verdigris River approximately four and one-half months after their release.

Application of Randomly Amplified Polymorphic DNA (RAPD) Fingerprinting to Detect Genetic Variation in Sericea Lespedeza (Lespedeza cuneata)

Abstract Sericea lespedeza (Lespedeza cuneata) is a Kansas statewide noxious weed that is spreading at an alarming rate. The genetic variation among different populations of this plant was investigated using the methodology of randomly amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR). Samples of sericea lespedeza were obtained at distinct sites throughout eastern Kansas and total DNA prepared from each. Four arbitrary primers were used individually in a RAPD-PCR and the combined amplification patterns used to generate a dendrogram. Sixteen plants from nine different populations were grouped into nine unique genotypic groupings using this method. These data indicate that significant genetic variation exists among the different populations of sericea lespedeza examined and that RAPD-PCR is a valid and reproducible means for the detection of these differences.

Genotyping by Randomly Amplified Polymorphic DNA is Influenced by the Method of DNA Preparation

Abstract Randomly amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) was performed on DNA templates prepared from thirty veterinary Salmonella typhimurium isolates. The templates were isolated by four different methods, each differing in their level of purity as determined by 260 nm/280 nm ratios. Dendrogram analysis of RAPD-PCR amplification patterns resulted in the organisms being placed into a range of 26-12 discrete groupings dependent on the DNA template isolation method used. Thus, DNA purity plays an important role in amplification patterns obtained by RAPD-PCR and investigators must consider this variable before making judgements on the genetic relatedness of a group of organisms.