An Adapted Optical Density-Based Microplate Assay for Characterizing Actinobacteriophage Infection.

Bacteriophages are a key part of natural environments, and they have a powerful ability to shape bacterial populations. To understand how individual phages interact with slow-growing bacterial hosts such as actinomycetes, an easy and reliable method for quantifying long-term bacterial growth in the presence of phages is needed. Spectrophotometric microplate readers allow for high-throughput repeated measurements, but incubating a small volume for an extended time can present technical challenges. This procedure adapts a standard 96-well microplate to allow for the co-culturing of phages and bacteria without sub-sampling for 96 h, with the bacterial growth recorded every 8 h using spectrophotometric absorbance values. These optical density values are analyzed using R to yield infection metrics, including the percent growth inhibition, relative virulence, and the Stacy-Ceballos index. The methods outlined here provide an effective way to conduct and analyze extended-duration microplate growth curve experiments and includes modifications to reduce evaporation and lid condensation. These protocols facilitate microplate-based assays of interactions between slow-growing bacterial hosts and their bacteriophages.

Complete Genome Sequences of Chop, DelRio, and GrandSlam, Three Gordonia Phages Isolated from Soil in Central Arkansas.

Chop, DelRio, and GrandSlam are phage with a Siphoviridae morphotype isolated from soil in Arkansas using the host Gordonia terrae 3612. All three are temperate, and their genomes share at least 96% nucleotide identity. These phage are assigned to cluster DI based on gene content similarity to other sequenced actinobacteriophage.

From genetics to biotechnology: Synthetic biology as a flexible course-embedded research experience.

The need for changing how science is taught and the expansion of undergraduate research experiences is essential to foster critical thinking in the Natural Sciences. Most faculty research programs only involve a small number of upper-level undergraduate students each semester. The course-based undergraduate research experience (CURE) model enables more students to take ownership over an independent project and experience authentic research. Further, by creating projects that fit into a curriculum's learning goals and student-oriented outcomes, departments help strengthen critical thinking skills in the classroom. Here, we report on the incorporation of a synthetic biology CURE into a mid-level cellular biology course and two advanced level genetics/molecular biology courses. Synthetic biology involves systematic engineering of novel organisms, such as bacteria and plants, to work as functional devices to solve problems in medicine, agriculture, and manufacturing. The value of synthetic biology and its ultimate utility as a teaching tool relies on reusable, standard genetic parts that can be interchanged using common genetic engineering principles. This Synthetic biology CURE effectively achieves five essential goals: (1) a sense of project ownership; (2) self-efficacy: mastery of a manageable number of techniques; (3) increased tolerance for obstacles through challenging research; (4) increased communication skills; and (5) a sense of belonging in a larger scientific community. Based upon our student assessment data, we demonstrate that this course-based synthetic biology laboratory engages students directly in an authentic research experience and models important elements of collaboration, discovery, iteration, and critical thinking.

Complete Genome Sequences of Four Putatively Antibiotic-Producing Bacteria Isolated from Soil in Arkansas, USA.

Soil bacteria can be a valuable source of antimicrobial compounds. Here, we report the complete genomes of four soil bacteria that were isolated by undergraduate microbiology students as part of a course-based research experience. These genomes were assembled using a hybrid approach combining paired-end Illumina reads with Oxford Nanopore Technologies MinION reads.

Genome Sequences of Four Cluster P Mycobacteriophages.

Four bacteriophages infecting Mycobacterium smegmatis mc2155 (three belonging to subcluster P1 and one belonging to subcluster P2) were isolated from soil and sequenced. All four phages are similar in the left arm of their genomes, but the P2 phage differs in the right arm. All four genomes contain features of temperate phages.

Comparative genomics of Cluster O mycobacteriophages.

Mycobacteriophages--viruses of mycobacterial hosts--are genetically diverse but morphologically are all classified in the Caudovirales with double-stranded DNA and tails. We describe here a group of five closely related mycobacteriophages--Corndog, Catdawg, Dylan, Firecracker, and YungJamal--designated as Cluster O with long flexible tails but with unusual prolate capsids. Proteomic analysis of phage Corndog particles, Catdawg particles, and Corndog-infected cells confirms expression of half of the predicted gene products and indicates a non-canonical mechanism for translation of the Corndog tape measure protein. Bioinformatic analysis identifies 8-9 strongly predicted SigA promoters and all five Cluster O genomes contain more than 30 copies of a 17 bp repeat sequence with dyad symmetry located throughout the genomes. Comparison of the Cluster O phages provides insights into phage genome evolution including the processes of gene flux by horizontal genetic exchange.

Impact of Entomophaga maimaiga (Entomophthorales: Entomophthoraceae) on outbreak gypsy moth populations (Lepidoptera: Erebidae): the role of weather.

The fungal pathogen Entomophaga maimaiga Humber, Shimazu, and Soper is prevalent in gypsy moth [Lymantria dispar (L.)] populations throughout North America. To understand how weather-related variables influence gypsy moth-E. maimaiga interactions in the field, we measured fungal infection rates at 12 sites in central Pennsylvania over 3 yr, concurrently measuring rainfall, soil moisture, humidity, and temperature. Fungal mortality was assessed using both field-collected larvae and laboratory-reared larvae caged on the forest floor. We found significant positive effects of moisture-related variables (rainfall, soil moisture, and relative humidity) on mortality due to fungal infection in both data sets, and significant negative effects of temperature on the mortality of field-collected larvae. Lack of a clear temperature relationship with the mortality of caged larvae may be attributable to differential initiation of infection by resting spores and conidia or to microclimate effects. These relationships may be helpful in understanding how gypsy moth dynamics vary across space and time, and in forecasting how the gypsy moth and fungus will interact as they move into warmer or drier areas, or new weather conditions occur due to climate change.

Emergent fungal entomopathogen does not alter density dependence in a viral competitor.

Population cycles in forest Lepidoptera often result from recurring density-dependent epizootics of entomopathogens. While these systems are typically dominated by a single pathogen species, insects are often infected by multiple pathogens, yet little is known how pathogens interact to affect host dynamics. The apparent invasion of northeastern North America by the fungal entomopathogen Entomophaga maimaiga some time prior to 1989 provides a unique opportunity to evaluate such interactions. Prior to the arrival of E. maimaga, the oscillatory dynamics of host gypsy moth, Lymantria dispar, populations were apparently driven by epizootics of a nucleopolyhedrovirus. Subsequent to its emergence, E. maimaiga has caused extensive mortality in host populations, but little is known about how it has altered multigenerational dynamics of the gypsy moth and its virus. Here we compared demographic data collected in gypsy moth populations prior to vs. after E. maimaiga's invasion. We found that the recently invading fungal pathogen virtually always causes greater levels of mortality in hosts than does the virus, but fungal mortality is largely density independent. Moreover, the presence of the fungus has apparently not altered the gypsy moth-virus density-dependent interactions that were shown to drive periodic oscillations in hosts before the arrival of the fungus.

Comparing two methods for quantifying soil-borne Entomophaga maimaiga resting spores.

To improve usability of methods for quantifying environmentally persistent entomophthoralean resting spores in soil, we modified and tested two methods using resting spores (azygospores) of the gypsy moth pathogen Entomophaga maimaiga. Both methods were effective for recovering resting spores at concentrations >100 resting spores/g dry soil. While a modification of a method originally described by Weseloh and Andreadis (2002) recovered more resting spores than a modified method based on Percoll density gradients, the ability to estimate true densities from counts was similar for both methods. Regression equations are provided for predicting true resting spore densities from counts, with R(2) values for both methods ≥0.90.

Variability in azygospore production among Entomophaga maimaiga isolates.

This study describes in vitro and in vivo azygospore production by nine isolates of Entomophaga maimaiga, a fungal pathogen of the gypsy moth, Lymantria dispar. The three E. maimaiga isolates that consistently produced azygospores in vitro were also strong producers of azygospores in vivo. However, two additional isolates that were strong azygospore producers in vivo did not produce azygospores in vitro. Isolates that produced azygospores in vitro produced both conidia and azygospores more frequently in vivo than isolates not producing azygospores in vitro. In vitro azygospore production varied over time as well as by isolate. After >2years of cold storage, while three isolates continued in vitro azygospore production, three isolates no longer produced azygospores in vitro.

Plant-mediated alteration of the peritrophic matrix and baculovirus infection in lepidopteran larvae.

The peritrophic matrix (PM) lines the midgut of most insects, providing protection to the midgut epithelial cells while permitting passage of nutrients and water. Herein, we provide evidence that plant-mediated alteration of the PM contributes to the well-documented inhibition of fatal infection by Autographa californica multiple nucleopolyhedrovirus (AcMNPV) of Heliothis virescens F. larvae fed cotton foliage. We examined the impact of the PM on pathogenesis using a viral construct expressing a reporter gene (AcMNPV-hsp70/lacZ) orally inoculated into larvae with either intact PMs or PMs disrupted by Trichoplusia ni granulovirus occlusion bodies containing enhancin, known to degrade insect intestinal mucin. Larvae possessing disrupted PMs displayed infection foci (lacZ signaling) earlier than those with intact PMs. We then examined PMs from larvae fed artificial diet or plant foliage using electron microscopy; foliage-fed larvae had significantly thicker PMs than diet-fed larvae. Moreover, mean PM width was inversely related to both the proportion of larvae with lacZ signaling at 18h post-inoculation and the final percentage mortality from virus. Thus, feeding on foliage altered PM structure, and these foliage-mediated changes reduced baculoviral efficacy. These data indicate that the PM is an important factor determining the success of an ingested pathogen in foliage-fed lepidopteran larvae.

Induction of systemic acquired resistance in cotton foliage does not adversely affect the performance of an entomopathogen.

Baculoviral efficacy against lepidopteran larvae is substantially impacted by the host plant. Here, we characterized how baculoviral pathogenicity to cotton-fed Heliothis virescens larvae is affected by induction of systemic acquired resistance (SAR). Numerous studies have shown that SAR induced by the plant elicitor benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) can protect against plant pathogens, but reports on the impacts of SAR on chewing herbivores or on natural enemies of herbivores are few. We found that BTH application significantly increased foliar peroxidase activity, condensed tannin levels, and total phenolic levels but did not alter dihydroxyphenolic levels. Consumption of BTH-treated foliage did not influence H. virescens pupal weight or larval mortality by the microbial control agent Autographa californica multiple nucleopolyhedrovirus any more than did consumption of untreated foliage. Thus, activation of SAR, although it did not protect the plant against a chewing herbivore, also did not reduce the effect of a natural enemy on a herbivore, indicating that SAR and microbial control agents may be compatible components of integrated pest management.