APPLICATION OF A TRANSPOSON FOOTPRINTING TECHNIQUE FOR RAPID IDENTIFICATION OF SALMONELLA TYPHIMURIUM TN5 MUTANTS REQUIRED FOR SURVIVAL UNDER DESICCATION STRESS CONDITIONS

Salmonella spp. are one of the foodborne pathogens that can be isolated in the environments of poultry houses and desiccation is a potential stress condition that can influence the survival of Salmonella spp. in this environment. In order to investigate the desiccation survival mechanism of Salmonella spp. the genome of S. typhimurium ATCC 14028 was screened for the genes potentially required for survival during desiccation using a novel method based on Tn5 mutagenesis previously developed in our laboratory. This method, termed transposon footprinting, simultaneously amplifies the Tn5-flanking sequences in a complex pool of the Tn5 mutants. As the length of the amplified DNA fragment should be unique for each distinct Tn5 mutant, the polymerase chain reaction (PCR) products separated on an agarose gel generate transposon footprints with each band in the footprint representing the corresponding Tn5 mutant. By comparing the transposon footprints from the pools of S. typhimurium Tn5 mutants before and after exposure to desiccation, Tn5 mutants that were not recovered after the selection were rapidly identified that would be easily isolated for further genetic analysis.     

A CHEMILUMINESCENCE BIOSENSOR COUPLED WITH IMMUNOMAGNETIC SEPARATION FOR RAPID DETECTION OF SALMONELLA TYPHIMURIUM

A chemiluminescence biosensor, using a fiber-optic-linked photometer and a data acquisition unit connected to a PC, was developed in conjunction with immunomagnetic separation for rapid detection of Salmonella Typhimurium. Magnetic microbeads coated with Anti-Salmonella antibodies and anti-Salmonella antibodies conjugated with horseradish peroxidase (HRP) were added to artificially-inoculated samples, and the immuno-reaction was completed in 60 min resulting in a sandwich complex. A magnetic field was applied to collect magnetic beads and the addition of luminol to HRP-conjugated antibodies resulted in a chemiluminescence reaction. The signal was collected through a fiber optic light guide, measured with a photometer, and recorded in the data acquisition unit. The minimum detection limit of the chemiluminescence biosensor for S. Typhimurium was 1.97 × 10³ CFU/mL and the range of the detectable signal was from 8.6 to 350 mV for cell numbers from 1.97 × 10³ to 1.97 × 10⁶ CFU/mL. Signal values for 10⁶ CFU/mL of S. Typhimurium were at least 97 and 394% higher than the corresponding values for S. enteritidis and four times the signal values for others including S. montevideo, S. california, S. heidlberg, and S. seftenberg, respectively. The biosensor response showed a significant difference (P < 0.05) between 10³ CFU/mL S. Typhimurium and 10⁶ CFU/mL of commonly-occurring bacteria in foods including Listeria monocytogenes, Pseudomonas aeruginosa, Citrobacter freundii, Campylobacter jejuni, Escherichia coli O157, and generic Escherichia coli. A regression equation, V = 0.0262 N ^5.7713, with R²= 0.9713 was obtained for the calibration curve over the detection range for S. Typhimurium. The whole procedure could be completed within 90 min.     

RAPID SCREENING METHOD TO DETECT POTENTIAL BIPARENTAL INHERITANCE OF PLASTID DNA AND RESULTS FOR OVER 200 ANGIOSPERM SPECIES

We have developed a diagnostic method to screen rapidly for plant species potentially capable of biparental inheritance of plastid DNA using the DNA fluorochrome 4′,6-diamidino-2-phenylindole (DAPI) in conjunction with epifluorescence microscopy. Pollen shed from 235 plant species (including about 50 of agronomic importance) representing 80 families were screened. Putative plastid DNA was detected in the generative and/or sperm cells of pollen from 26 genera (43 species) representing 15 families. Plastid DNA was not detected in the generative or sperm cells of pollen from 192 plant species, thereby strongly suggesting that these species have only maternal inheritance. Our cytological diagnosis corroborated the known genetic evidence in 42 plant species and conflicted with the genetic reports in five species, which are discussed. The data suggest that biparental inheritance of plastids is rare; overall, it may occur in about 14% of flowering plant genera, examples of which are scattered among 19% of the families examined. This methodology also readily reveals whether pollen is bi- or trinucleate.     

THE POTENTIAL FOR COEVOLUTION IN A HOST-PARASITOID SYSTEM. II. GENETIC VARIATION WITHIN A POPULATION OF WASPS IN THE ABILITY TO PARASITIZE AN APHID HOST

Much of the study of coevolution has focused on the adaptations that have resulted from interactions between species. For reciprocal evolution to occur, there must be genetic variation in each species for traits that directly affect their interaction. Here I report evidence of significant additive genetic variance within a population of parasitic wasps in the ability to successfully parasitize an aphid host. These data, combined with companion work documenting clonal variation in a population of aphids from the same site, provide evidence that within the same population both a host and its parasitoid have the potential for specific and reciprocal genetic interactions.