Bacteriophage MooMoo is a temperate phage that was isolated and propagated on Mycobacterium smegmatis (M. smeg). It typically produces turbid plaques, however spontaneous clear plaque mutants can be readily isolated. Both turbid (MooMoo-T) and clear plaque (MooMoo-C) formers can establish stable lysogens, but the parental turbid plaque forming phage has a higher lysogenic frequency. The phage repressor protein typically plays the central role in regulating the lysis/lysogeny decision. Therefore, we expected that the mutation responsible for the clear plaque phenotype would be located in the repressor gene. Remarkably, whole genome sequencing detected a single base pair mutation in the minor tail protein gene (gp19). The regulatory role of the repressor protein could not be excluded considering it was unclear how the mutation in gp19 was leading to the altered plaque phenotype. To locate the phage repressor, we used bioinformatics to identify several candidate genes with helix-turn-helix and DNA binding motifs (gp42, gp43 and gp44). We also cloned the parental and mutant gp19 genes. Each candidate gene was cloned into a shuttle vector. The clones of gp43, gp44 and both derivatives of gp19 did not prevent MooMoo growth, whereas the clones of gp42 inhibited phage growth. Based on these results, we concluded that gp42 is the phage repressor for MooMoo. To determine if the presence of gp19 alters lysogenic frequency, lysogeny assays of wild-type (WT) and mutant gp19 clones were evaluated. Compared to the MooMoo-C lysate, the cloned copy of the mutant gp19 showed a slight increase in lysogeny efficiency. The lysogeny frequencies on strains that carry cloned copies of gp19 (WT or mutant) were similar to those obtained on strains that lacked the plasmids. From these results, we concluded, the presence of either parental or mutant gp19 clones does not affect the lysogeny frequency. To determine if host cell physiology was affected by lysogeny, carbon, nitrogen, phosphorus and sulfur utilization resources were screened using high-throughput phenotypic microarrays. From these results, we concluded the presence of the WT or mutant prophage had no significant effect on the utilization of the resources tested.

Phytophthora cinnamomi is a soil-borne oomycete pathogen causing root rot in susceptible host species. P. cinnamomi is thought to have originated in Southeast Asia, but has since been introduced to many regions around the world, where it causes dramatic declines in many forest tree species. In the eastern US, the primary susceptible tree species of concern are American chestnut (Castanea dentata), white oak (Quercus alba), and shortleaf pine (Pinus echinata). American chestnut, functionally eliminated in the early 1900s by the rapidly acting chestnut blight (Cryphonectria parasitica), has been the subject of decades-long breeding efforts aimed at improving chestnut resistance to chestnut blight. To improve chestnut restoration success, and restoration of other susceptible species, the distribution patterns of P. cinnamomi on a landscape scale must be better understood. This project was initiated to develop an improved method for detecting P. cinnamomi to permit high-throughput screening of forest soils, and to implement the improved detection approach in characterizing the distribution patterns of P. cinnamomi in developing soils on reclaimed surface mines in eastern Kentucky, as well as mature forest soils within an undisturbed watershed in a reference-quality eastern Kentucky forest. We developed an improved detection method using a molecular DNA-amplification approach (PCR), which demonstrated similar sensitivity to traditional culture-based methods, but required less time and space than traditional methods. We used this detection approach to screen soils from a chronosequence of reclaimed surface mines (reclaimed at different points in time) to evaluate whether reclaimed surface mined sites become favorable for P. cinnamomi colonization over time. Our analysis detected P. cinnamomi at the two older sites (reclaimed in 1997 and 2003), but we did not detect P. cinnamomi at the two newer sites sampled (reclaimed in 2005 and 2007). These results suggest that surface mined sites become favorable for P. cinnamomi colonization over time, and should not be considered permanently “Phytophthora-free.” We also collected ~200 samples from a watershed in UK’s Robinson Forest, from plots representing a gradient of topographic position, slope, and aspect. This survey indicated that P. cinnamomi distribution in forests is complex and can be difficult to predict; however, P. cinnamomi was detected in both drier upslope sites and in moister drainage sites.

Agricultural intensification reliant on monocrops could change soil health in a way that does not support maximum crop productivity. Twenty-nine-year-old no-till field plots at the University of Kentucky Spindletop research farm showed a significant reduction in corn yields from continuous corn plots compared to those from plots in various types of rotation. The objective of this study was to determine what role soil microbes might play in yield reduction and how management and time effects microbial community structure. Samples were collected from the following treatments: continuous corn (CC), continuous soybean (SS), a 2-year corn/soybean rotation (CCSS), Corn in rotation with soybean with winter wheat cover (C/W/S), and sod controls (SOD). Soil health-related parameters were determined along with microbial community structure using phospholipid fatty acid analysis (PLFA). Results show that there is a strong seasonal dynamic in microbial communities with May, July and September showing the greatest differentiation between treatments. Nonparametric multidimensional analysis (NMDS) shows that microbial communities under SS, CC treatments were significantly different from the CS and CWS treatments across all four years of the study. My findings will prove useful for assessing the contribution of biological indicators to agroecosystem function and will aid in making recommendations of when and how to manage these parameters to improve soil health and maximize yield.

Atribacteria (OP9), candidate phylum with no representatives in pure culture, is found in various anaerobic environments worldwide. “Caldatribacterium”, a lineage within Atribacteria that is predicted to be a strictly anaerobic sugar fermenter based on cultivation-independent genomic analyses, is currently being maintained in lab enrichment cultures with fucose as its sole growth substrate. Metagenomics and 16S rRNA gene tag sequencing indicated that the fucose culture was a co-culture of “Caldatribacterium” and an uncultivated member of the genus Thermodesulfobacterium. Due to failed attempts to isolate “Caldatribacterium” by dilution-to-extinction and plating, it was hypothesized that “Caldatribacterium” is dependent in some way on the Thermodesulfobacterium. To better understand the possible interaction, multiple isolates of the sulfate reducer were obtained under sulfate-reducing conditions with H2 as an electron donor, and one of the isolates was characterized. Whole genome and 16S rRNA gene sequence comparisons of the isolate and other related members of the genus Thermodesulfobacterium suggested the isolate represents a distinct species in this genus, for which the name T. auxiliatoris is proposed. T. auxiliatoris was capable of using H2, formate, and lactate as sole electron donors, but not fucose or other sugars, suggesting that its growth in the co-culture might be dependent on one or more fermentation substrates produced by “Caldatribacterium”. Addition of T. auxiliatoris to highly diluted samples of the co-culture that likely contained only “Caldatribacterium”, which did not exhibit growth on their own, demonstrated that T. auxiliatoris was sufficient to support growth of “Caldatribacterium” on fucose. When this dilution experiment was repeated with various other organisms and substrates, it was found that several other thermophilic sulfate reducers (T. commune, T. hveragerdense, or Thermodesulfovibrio yellowstonii) could also support growth, as well as supernatant from the T. auxiliatoris pure culture or yeast extract. This last finding allowed for isolation of “Caldatribacterium”, which could form colonies on solid media when yeast extract and casamino acids were present. Fluorescent in situ hybridization and nanometer-scale secondary ion mass spectrometry demonstrated that “Caldatribacterium” took up a variety of sugars and amino acids in mixed culture, and that addition of acetate or bicarbonate, substrates of T. auxiliatoris, stimulated sugar uptake in “Caldatribacterium”. These results support a model where T. auxiliatoris and “Caldateribacterium” are dependent on each other in co-culture on fucose, where “Caldatribacterium” provides growth substrates for T. auxiliatoris, which in turn provides “Caldatribacterium” with some sort of soluble, essential compound(s) that can be produced by other sulfate reducers and are present in yeast extract. Further characterization of the “Caldatribacterium” isolate, the first representative of the phylum Atribacteria, will allow for detailed study of its metabolic capabilities that can be extended to other members of this phylum. Further analysis of responses of T. auxiliatoris and “Caldatribacteirum” when grow in co-culture and the specific metabolite(s) that are exchanged between the two organisms could allow for testing whether these interactions occur in more complex, natural systems.

The unicellular cyanobacterium Prochlorococcus is the numerically dominant photosynthetic organism throughout the temperate and tropical open oceans, but it is difficult to grow in pure cultures. We developed a system for rendering spontaneous streptomycin-resistant mutants of Prochlorococcus axenic by diluting them to extinction in the presence of “helper” heterotrophic bacteria, allowing them to grow to high cell concentrations, and then killing the helpers with streptomycin. Using axenic strains obtained in this fashion, we demonstrated that Prochlorococcus experiences a number of growth defects in dilute axenic culture, including reduced growth rate, inability to form colonies on solid media, and higher incidence of mortality (i.e., catastrophic failure of liquid cultures). These defects were eliminated when Prochlorococcus was grown in co-culture with a phylogenetically diverse array of helper bacteria. The primary mechanism of helping was enzymatic removal of hydrogen peroxide (HOOH) from the culture medium. Axenic Prochlorococcus cultures were profoundly sensitive to HOOH additions in comparison with reported tolerance levels for all other wild-type aerobic bacteria, but in co-culture their resistance was similar to that of the helpers. Neither is dependence on helpers limited to the laboratory. Sterile-filtered seawater exposed to sunlight accumulated enough HOOH in 24h to kill ecologically relevant cell concentrations of Prochlorococcus. We also refined a method for delivering HOOH at a defined, steady rate using the buffer HEPES to more accurately simulate the steady accumulation of HOOH in natural waters. Even at the lowest production rates that could sustain the in situ HOOH concentration in the ocean, HEPES-generated HOOH was lethal to Prochlorococcus; again, co-culture with helpers prevented this effect. We speculate on the ecological consequences of Prochlorococcus’ dependency on other organisms for survival, as well as the evolutionary forces that have led to this lack of self-sufficiency.

A recent study using human fecal samples in conjunction with a culture-independent approach revealed immense diversity of antibiotic resistance (AR) genes in the human gut microflora. We hypothesize that food animal gut microflora also contain diverse and novel AR genes which could contribute to the emergence and transmission of AR in pathogens important in animal and human health. To test this, we examined AR reservoir in chicken gut microflora using a metagenomic, functional cloning method. Total genomic DNA was extracted from individual cecal contents of two free range chickens and two conventionally raised chickens. The DNAs were physically sheered into 1 to 3 kb fragments, cloned into expression vector pZE21-MCS, and transformed into E. coli TOP10 host strain, resulting in four metagenomic libraries of a total size of 108 base pairs per library. The AR transformants from the libraries were selected on plates containing the specific antibiotic of interest; six antibiotics including ampicillin, tetracycline, chloramphenicol, spectinomycin, ciprofloxacin and norfloxacin were used for screening. Plasmids from selected transformants were extracted and subjected to sequence analysis of inserted fragments. Identified AR genes were annotated and aligned with homologs that have been deposited in GenBank. A total of 12 AR genes and 3 AR genes were identified from the microbiome in conventionally raised chickens and free-range chickens, respectively. Of the identified 15 AR genes, 8 genes that confer resistance to ampicillin, spectinomycin or chloramphenicol shared low sequence similarity (58% - 76% at amino acid level) with the corresponding AR genes previously identified using culture-dependent approaches. Notably, among the 8 novel AR genes identified in this study, 4 genes also shared low sequence similarities (59%-76% at amino acid level) with recently identified AR genes in human gut. An E. coli-Campylobacter shuttle vector bearing the flaA sigma 28 promoter was constructed. Two novel genes conferring resistance to ampicillin (FRAmp1.1) and spectinomycin (FRSpe1.1) were cloned into this new expression vector, respectively. The derived vectors have conferred increased AR in C. jejuni, a leading zoonotic bacterial pathogen causing human gastroenteritidis in many industrialized countries. Together, findings from this study showed the effectiveness of the metagenomic approach for examination of AR reservoir in food animals, revealed novel AR resistance genes in chicken gut microflora, and demonstrated the functionality of such AR genes in foodborne human pathogens.

Climate change is likely to alter plant species composition and interactions between plants and soil microbes that together dictate the quantity and quality of forage produced in pastures, the base of animal production in central Kentucky. This study assessed the seasonal dynamics of soil microbes and their response to increased temperature (+3oC) and growing season precipitation (+30% of the mean annual). Total soil microbial biomass, community composition, enzyme activities, potential carbon mineralization, and catabolic responses to selected substrates were measured seasonally in the different climate treatments. In this system, seasonal variability was a dominant driving factor for all the soil microbial characteristics that I investigated. Summer maxima and winter minima were identified in the active microbial biomass, while soil microbial community structure differed between each season. Extracellular enzyme activities were generally highest in either the spring or summer, while seasonal patterns for each substrate were unique across catabolic response profiles. Climate treatments produced few significant main or interactive effects on the soil microbial biomass and function. This resiliency, coupled with evidence of functional redundancy, suggests that central Kentucky pasture ecosystems may be well-equipped to handle future environmental stress associated with climate change and to maintain critical ecosystem services.

Runoff from livestock operations may contain a variety of pathogens and high levels of nutrients and other harmful contaminants, and is of particular concern in central Kentucky as watersheds are threatened by waste generated from a high concentration of equine activity. Rain gardens are a type of stormwater management tool used to capture and passively treat runoff. This project aimed to incorporate rain gardens into the horse muck storage structures at a thoroughbred facility in the Canr Run watershed in Lexington, Kentucky. Water quality data from soil water within two rain garden muck pads and two control pads, and grab samples from the stream were compared. No significant differences were observed, but trends revealed higher levels of nitrate and phosphate in rain gardens compared to controls, while total organic carbon and E. coli levels were lower in the rain gardens, suggesting that the rain gardens are trapping nutrients while reducing organic matter and killing bacteria. E. coli populations were lower in stream sample locations near rain garden muck pads compared to further downstream near controls. Management recommendations include further improvement of muck storage structures, replacing old muck pads, and changing management and housekeeping habits and attitudes towards environmental responsibility.

Manufactured CeO2 nanomaterials (CeO2-MNMs) are used for a wide variety of applications including diesel fuel additives and chemical/mechanical planarization media. To test the effects of CeO2-MNM surface coating charge on to model organism Caenorhabditis elegans, we synthesized 4 nm CeO2 with cationic (DEAE-), anionic (CM-), and neutral (DEX) coatings. In L3 nematodes exposed for 24 hours, DEAE-CeO2 induced lethality at lower concentrations than CM- or DEX-CeO2. Feeding slightly decreased CeO2 toxicity, regardless of coating. In L2 nematodes exposed for 48 hours with feeding, DEAE-CeO2 caused lethality at the lower concentrations as compared to CM- and DEX-CeO2. Sublethal effects were measured by observing reproduction and oxidative/nitrosative protein damage. Low concentrations of DEAE-CeO2 induced similar reductions as CM- and DEX-CeO2 that were two orders of magnitude higher. Using immunochemical slot blots to explore oxidative/nitrosative stress, no treatments produced significant changes in protein carbonyl or 3-nitrotyrosine formation; however, the statistical power of our assay was low. All treatments caused large but not statistically significant increases in protein carbonyl levels. DEAE-CeO2 exposure caused a significant reduction in 4-hydroxy-2-nonenol levels. This research suggests that cationic coatings render CeO2 significantly more toxic to C. elegans than neutral or anionic coatings.

To gain a better understanding of contaminating bacteria in bioethanol industry, we profiled the bacterial community structure in corn-based bioethanol fermentations and evaluated its correlation to environmental variables. Twenty-three batches of corn-mash sample were collected from six bioethanol facilities. The V4 region of the collective bacterial 16S rRNA genes was analyzed by Illumina Miseq sequencing to investigate the bacterial community structure. Non-metric multidimensional scaling (NMDS) ordination plots were constructed to visualize bacterial community structure groupings among different samples, as well as the effects of multiple environmental variables on community structure variation. Our results suggest that bacterial community structure is facility-specific, although there are two core bacterial phyla, Firmicutes and Proteobacteria. Feedstock, facility, and fermentation technology may explain the difference in community structure between different facilities. Lactic acid, the most important environmental variable that influences bacterial community structure grouping, could be utilized as an indicator of bacterial contamination. We also identified genes responsible for the multiple antibiotic-resistance phenotype of an Enterobacter cloacae strain isolated from a bioethanol fermentation facility. We performed PCR assays and revealed the presence of canonical genes encoding resistance to penicillin and erythromycin. However, a gene encoding resistance to virginiamycin was not detected.