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Research Assistant Professor
PhD Marine Biosciences, University of Delaware, 2007
MS Applied Molecular Biology, University of Maryland, 1993
BS Biological Sciences, University of Maryland, 1991
Dr. Waidner’s current research interests are in environmental microbiology, microbial ecology, and bioremediation in oceans, coastal waters, inland bays, and rivers.
- A MicroRNA of infectious laryngotracheitis virus can downregulate and direct...
- Viral microRNAs regulate gene expression using either translational repression or mRNA cleavage and decay. Two microRNAs from infectious laryngotracheitis virus (ILTV), iltv-miR-I5 and iltv-miR-I6, map antisense to the ICP4 gene. Post-transcriptional repression by these microRNAs was tested against a portion of the ICP4 coding sequence cloned downstream of firefly luciferase. Luciferase activity was downregulated by approximately 60% with the iltv-miR-I5 mimic. Addition of an iltv-miR-I5 antagomiR or mutagenesis of the target seed sequence alleviated this effect. The iltv-miR-I5 mimic, when co-transfected with a plasmid expressing ICP4, reduced ICP4 transcript levels by approximately 50%, and inhibition was relieved by an iltvmiR- I5 antagomiR. In infected cells, iltv-miR-I5 mediated cleavage at the canonical site, as indicated by modified RACE analysis. Thus, in this system, iltv-miR-I5 decreased ILTV ICP4 mRNA levels via transcript cleavage and degradation. Downregulation of ICP4 could impact the balance between the lytic and latent states of the virus in vivo., Journal Article, Author's copy
- Abundance, diversity, and distribution of aerobic anoxygenic phototrophic bacteria...
- Several groups of bacteria have the capacity to derive extra energy from light while still oxidizing organic matter for carbon and energy. Among these photoheterotrophs are the aerobic anoxygenic photosynthetic (AAP) bacteria, which harvest light energy with the use of bacteriochlorophyll a but do not evolve oxygen. The goals of this dissertation work were to determine ecological controls on the distribution of AAP bacteria in estuaries. In eight transects over five years, AAP abundance was measured in the Delaware and Chesapeake estuaries. Both microscopy and quantitative PCR (qPCR) data indicated that AAP bacteria comprised a large portion of the prokaryotic community in the estuaries examined. In the Delaware, AAP bacteria were on average 12% of bacteria, while in the Chesapeake they were less abundant, making up on average 6% of cells. As determined by qPCR, 0.6 to 50% of bacteria were AAP in the Delaware. In both estuaries, AAP abundance was inversely correlated with light, indicating they were particularly abundant in turbid waters. These results were surprising, since the photoheterotrophic capabilities of these bacteria should provide them with competitive advantages in environments with high light availability. To further examine the ecological implications of turbidity, I tested for AAP particle-attachment preferences. Up to 90% of AAP bacteria were attached to particles, while usually 20% or less of the total prokaryotic community was in the particle-attached fraction. By both microscopy and qPCR, it was determined that particle-attached AAP bacterial abundance estimates were positively correlated with seston concentrations (r=0.65, p<0.01 and r=0.68, p<0.01, respectively). These results suggest that estuarine types of AAP bacteria are particularly competitive in high-nutrient, turbid waters. To examine the potential ecological controls over subsets of the AAP bacterial community, I first examined metagenomic and PCR libraries for diversity of pufM, a photosynthesis reaction center gene common to all AAP bacteria. The metagenomic data revealed the presence of two distinct types of AAP bacteria in the Delaware River. Extensive phylogenetic analyses of light-harvesting and non-phototrophy genes suggested that the two types of AAP in the metagenomic data set occupy two distinct niches (freshwater and estuarine) in estuaries. To further examine the diversity of freshwater and estuarine types of AAP bacteria, I amplified the pufM genes from microbial consortia DNA harvested from the Delaware River. In two pufM libraries constructed from summer and winter samples, the most prevalent form of pufM was the freshwater ecotype, but the estuarine type of this gene was only a minor member of the AAP bacterial community. However, in a study of the actively expressed pufM genes in the river, the estuarine type was more important in the community, comprising 10% of the clones. The data from the metagenomic study and the PCR diversity study led to my hypothesis that two niches would be occupied by AAP bacteria containing these different types of genes. qPCR assays were then designed to target freshwater, estuarine, and marine types of AAP bacteria of the Delaware estuary, and the physical distribution of these three ecotypes were examined in four transects of the estuary in 2002-2004. In all samplings, both the freshwater and marine types were most abundant in the riverine and bay ends of the estuary, declining with water mixing. The estuarine type, however, did not follow consistent patterns. In summer, this type of pufM was more abundant in the bay, while it was shifted upstream into the freshwaters in the autumn and winter samples. Finally, to examine the particle-attached and free-living preferences among these three ecotypes of AAP bacteria, I examined their abundances in two size fractions. In the free-living fraction, all three ecotypes followed distribution patterns similar to those of the 2002-2004 transects. In contrast, the marine, freshwater, and estuarine ecotypes in whole water were most abundant in mid-estuary near the turbidity maximum. These data suggest that particle-attachment is a general phenomenon among dominant groups of AAP in the estuary. Overall, the data in this dissertation provide clues to the environmental adaptations of estuarine AAP bacteria, but more diversity and distribution studies are needed to further understand the ecological controls of AAP bacteria., UMI Number: 3267188
- Abundant proteorhodopsin genes in the North Atlantic Ocean
- Proteorhodopsin (PR) is a light-driven proton pump that has been found in a variety of marine bacteria, including Pelagibacter ubique, a member of the ubiquitous SAR11 clade. The goals of this study were to explore the diversity of PR genes and to estimate their abundance in the North Atlantic Ocean using quantitative polymerase chain reaction (QPCR). We found that PR genes in the western portion of the Sargasso Sea could be grouped into 27 clusters, but five clades had the most sequences. Sets of specific QPCR primers were designed to examine the abundance of PR genes in the following four of the five clades: SAR11 (P. ubique and other SAR11 Alphaproteobacteria), BACRED17H8 (Alphaproteobacteria), HOT2C01 (Alphaproteobacteria) and an uncultured subgroup of the Flavobacteria. Two groups (SAR11 and HOT2C01) dominated PR gene abundance in oligotrophic waters, but were significantly less abundant in nutrient- and chlorophyll-rich waters. The other two groups (BACRED17H8 and Flavobacteria subgroup NASB) were less abundant in all waters. Together, these four PR gene types were found in 50% of all bacteria in the Sargasso Sea. We found a significant negative correlation between total PR gene abundance and nutrients and chlorophyll but no significant correlation with light intensity for three of the four PR types in the depth profiles north of the Sargasso Sea. Our data suggest that PR is common in the North Atlantic Ocean, especially in SAR11 bacteria and another marine alphaproteobacterial group (HOT2C01), and that these PR-bearing bacteria are most abundant in oligotrophic waters., Journal Article, Final article published
- Aerobic anoxygenic photosynthesis genes and operons in uncultured bacteria in...
- Photosynthesis genes and operons of aerobic anoxygenic photosynthetic (AAP) bacteria have been examined in a variety of marine habitats, but genomic information about freshwater AAP bacteria is lacking. The goal of this study was to examine photosynthesis genes of AAP bacteria in the Delaware River. In a fosmid library, we found two clones bearing photosynthesis gene clusters with unique gene content and organization. Both clones contained 37 open reading frames, with most of those genes encoding known AAP bacterial proteins. The genes in one fosmid were most closely related to those of AAP bacteria in the Rhodobacter genus. The genes of the other clone were related to those of freshwater beta-proteobacteria. Both clones contained the acs F gene, which is required for aerobic bacteriochlorophyll synthesis, suggesting that these bacteria are not anaerobes. The beta-proteobacterial fosmid has the puf operon B-AL-M-C and is the first example of an uncultured bacterium with this operon structure. The alpha-3-proteobacterial fosmid has a rare gene order (Q-B-A-L-M-X), previously observed only in the Rhodobacter genus. Phylogenetic analyses of photosynthesis genes revealed a possible freshwater cluster of AAP betaproteobacteria. The data from both Delaware River clones suggest there are groups of freshwater or estuarine AAP bacteria distinct from those found in marine environments., Journal Article, Final article published
- Aerobic anoxygenic phototrophic bacteria attached to particles in turbid waters...
- Aerobic anoxygenic phototrophic (AAP) bacteria are photoheterotrophs that, if abundant, may be biogeochemically important in the oceans. We used epifluorescence microscopy and quantitative PCR (qPCR) to examine the abundance of these bacteria by enumerating cells with bacteriochlorophyll a (bChl a) and the light-reaction center gene pufM, respectively. In the surface waters of the Delaware estuary, AAP bacteria were abundant, comprising up to 34% of prokaryotes, although the percentage varied greatly with location and season. On average, AAP bacteria made up 12% of the community as measured by microscopy and 17% by qPCR. In the surface waters of the Chesapeake, AAP bacteria were less abundant, averaging 6% of prokaryotes. AAP bacterial abundance was significantly correlated with light attenuation (r = 0.50) and ammonium (r = 0.42) and nitrate (r = 0.71) concentrations. Often, bChl a-containing bacteria were mostly attached to particles (31 to 94% of total AAP bacteria), while usually 20% or less of total prokaryotes were associated with particles. Of the cells containing pufM, up to 87% were associated with particles, but the overall average of particleattached cells was 15%. These data suggest that AAP bacteria are particularly competitive in these two estuaries, in part due to attachment to particles., Journal Article, Final article
- Bacterial diversity of metagenomic and PCR libraries from the Delaware River...
- To determine whether metagenomic libraries sample adequately the dominant bacteria in aquatic environments, we examined the phylogenetic make-up of a large insert metagenomic library constructed with bacterial DNA from the Delaware River, a polymerase chain reaction (PCR) library of 16S rRNA genes, and community structure determined by fluorescence in situ hybridization (FISH). The composition of the libraries and community structure determined by FISH differed for the major bacterial groups in the river, which included Actinobacteria, beta-proteobacteria and Cytophaga-like bacteria. Beta-proteobacteria were underrepresented in the metagenomic library compared with the PCR library and FISH, while Cytophaga-like bacteria were more abundant in the metagenomic library than in the PCR library and in the actual community according to FISH. The Delaware River libraries contained bacteria belonging to several widespread freshwater clusters, including clusters of Polynucleobacter necessarius, Rhodoferax sp. Bal47 and LD28 beta-proteobacteria, the ACK-m1 and STA2-30 clusters of Actinobacteria, and the PRD01a001B Cytophaga-like bacteria cluster. Coverage of bacteria with > 97% sequence identity was 65% and 50% for the metagenomic and PCR libraries respectively. Rarefaction analysis of replicate PCR libraries and of a library constructed with re-conditioned amplicons indicated that heteroduplex formation did not substantially impact the composition of the PCR library. This study suggests that although it may miss some bacterial groups, the metagenomic approach can sample other groups (e.g. Cytophaga-like bacteria) that are potentially underrepresented by other culture-independent approaches., Journal Article, Final article published
- Diversity and abundance of glycosyl hydrolase family 5 in the North Atlantic...
- The diversity and abundance of glycosyl hydrolase family 5 (GH5) were studied in the North Atlantic Ocean. This family was chosen because of the large number of available sequences from cultured bacteria, the variety of substrates it targets, and the high number of similar sequences in the Sargasso Sea environmental genome database. Three clone libraries of a GH5 subcluster were constructed from the Mid-Atlantic Bight and the eastern and western North Atlantic Ocean. The two North Atlantic Ocean libraries did not differ from each other but both were significantly less diverse than the Mid-Atlantic Bight library. The abundance of GH5 genes estimated by quantitative PCR was positively correlated with chlorophyll concentrations in the eastern part of a transect from Fort Pierce, Florida, to the Azores and in a depth profile, suggesting that the supply of labile organic material selects for GH5-bearing bacteria in these waters. However, the data suggest that only o1% of all bacteria harbor the GH5 subcluster. These and other data suggest that the hydrolysis of polysaccharides requires complicated multienzyme systems., Journal Article, Final article published
- Diversity and distribution of ecotypes of the aerobic anoxygenic phototrophy...
- The diversity of aerobic anoxygenic phototrophic (AAP) bacteria has been examined in marine habitats, but the types of AAP bacteria in estuarine waters and distribution of ecotypes in any environment are not well known. The goal of this study was to determine the diversity of AAP bacteria in the Delaware estuary and to examine the distribution of select ecotypes using quantitative PCR (qPCR) assays for the pufM gene, which encodes a protein in the light reaction center of AAP bacteria. In PCR libraries from the Delaware River, pufM genes similar to those from Beta- (Rhodoferax-like) or Gammaproteobacteria comprised at least 50% of the clones, but the expressed pufM genes from the river were not dominated by these two groups in August 2002 (less than 31% of clones). In four transects, qPCR data indicated that the gammaproteobacterial type of pufM was abundant only near the mouth of the bay whereas Rhodoferax-like AAP bacteria were restricted to waters with a salinity of <5. In contrast, a Rhodobacter-like pufM gene was ubiquitous, but its distribution along the salinity gradient varied with the season. High fractions (12 to 24%) of all three pufM types were associated with particles. The data suggest that different groups of AAP bacteria are controlled by different environmental factors, which may explain current difficulties in predicting the distribution of total AAP bacteria in aquatic environments., Journal Article, Final article
- Domain effects on the DNA-interactive properties of bacteriophage T4 gene 32...
- Bacteriophage T4 gene 32 protein, a model for singlestrand specific nucleic acid-binding proteins, consists of three structurally and functionally distinct domains. We have studied the effects of the N and C domains on the protein structure and its nucleic acid-interactive properties. Although the presence of the C domain decreases the proteolytic susceptibility of the core (central) domain, quenching of the core tryptophan fluorescence by iodide is unaltered by the presence of the terminal domains. These results suggest that the overall conformation of the core domain remains largely independent of the flanking domains. Removal of the N or the C terminus does not abolish the DNA renaturation activity of the protein. However, intact protein and its three truncated forms differ in DNA helix-destabilizing activity. The C domain alone is responsible for the kinetic barrier to natural DNA helix destabilization seen with intact protein. Intact protein and core domain potentiate the DNA helix-destabilizing activity of truncated protein lacking only the C domain (*I), enhancing the observed hyperchromicity while increasing the melting temperature. Proteolysis experiments suggest that the affinity of core domain for single-stranded DNA is increased in the presence of *I. We propose that *I can “mingle” with intact protein or core domain while bound to single-stranded DNA., Journal Article, Final article published
- Evaluation of two approaches for assessing the genetic similarity of virioplankton...
- Viral production estimates show that virioplankton communities turn over rapidly in aquatic ecosystems. Thus, it is likely that the genetic identity of viral populations comprising the virioplankton also change over temporal and spatial scales, reflecting shifts in viral-host interactions. However, there are few approaches that can provide data on the genotypic identity of viral populations at low cost and with the sample throughput necessary to assess dynamic changes in the virioplankton. This study examined two of these approaches-T4-like major capsid protein (g23) gene polymorphism and randomly amplified polymorphic DNA-PCR (RAPD-PCR) fingerprinting-to ask how well each technique could track differences in virioplankton populations over time and geographic location. Seasonal changes in overall virioplankton composition were apparent from pulsed-field gel electrophoresis (PFGE) analysis. T4-like phages containing similar g23 proteins were found within both small- and large-genome populations, including populations from different geographic locations and times. The surprising occurrence of T4-like g23 within small genomic groups (23 to 64 kb) indicated that the genome size range of T4-like phages may be broader than previously believed. In contrast, RAPD-PCR fingerprinting detected high genotypic similarity within PFGE bands from the same location, time, and genome size class without the requirement for DNA sequencing. Unlike g23 polymorphism, RAPD-PCR fingerprints showed a greater temporal than geographic variation. Thus, while polymorphism in a viral signature gene, such as g23, can be a powerful tool for inferring evolutionary relationships, the degree to which this approach can capture fine-scale variability within virioplankton populations is less clear., We acknowledge the support of National Science Foundation grants (EF-0626826, MCB-0132070, and MCB-0731916) to K.E.W. We are indebted to the crew of the R/V Cape Henlopen and Hugh R. Sharp for their tireless assistance in the field., Journal Article, Final article published
- MicroRNAs of gallid and meleagrid herpesviruses show generally conserved genomic...
- Many herpesviruses, including Marek's disease viruses (MDV1 and MDV2), encode microRNAs. In this study,we report microRNAs of two related herpesviruses, infectious laryngotracheitis virus (ILTV) and herpesvirus of turkeys (HVT), as well as additional MDV2 microRNAs. The genome locations, but not microRNA sequences, are conserved among all four of these avian herpesviruses. Most are clustered in the repeats flanking the unique long region (I/TRL), except in ILTV which lacks these repeats. Two abundant ILTV microRNAs are antisense to the immediate early gene ICP4. A homologue of host microRNA, gga-miR-221, was found among the HVT microRNAs. Additionally, a cluster of HVT microRNAs was found in a region containing two locally duplicated segments, resulting in paralogous HVT microRNAs with 96– 100% identity. The prevalence of microRNAs in the genomic repeat regions as well as in local repeats suggests the importance of genetic plasticity in herpesviruses for microRNA evolution and preservation of function., Journal Article, Final article published
- Return of the native: Historical comparisons of invasive and indigenous crab...
- An invasive population of the Asian shore crab Hemigrapsus sanguineus was discovered in 1988 near the mouth of Delaware Bay, and populations now occur from North Carolina to Maine. The shore crab H. sanguineus competes with indigenous species and has displaced resident crabs throughout its invasive range. However, there have been few studies that document changes in populations of H. sanguineus after the species has become established.We compare sympatric populations of the Asian shore crab and a native mud crab (Panopeus herbstii) that were monitored initially in 2001 and again in 2011 and 2012. The historical study was conducted in a rocky habitat near Cape Henlopen at the southern terminus of Delaware Bay (38.793° N, 75.158° W). Results showed large differences in the relative abundance of the two species throughout the duration of the study. The Asian shore crab H. sanguineus accounted for 75% of total crab abundance in 2001, but abundance had decreased to less than 25% in both 2011 and 2012. Similar results were obtained when we compared the two species in terms of biomass. Additional sampling in 2012 showed comparable low values for H. sanguineus when compared with P. herbstii at two stations about 25 km and 50 km farther south along the coast. In contrast, H. sanguineus was strongly dominant at a station 50 km north of the historical sampling site. Percentage rock cover and size of rocks varied little among sampling locations, and all sites were proximal to the coastal ocean. However, basal sediment at the northern station was coarser than sediments at the other sites, which may have restricted the occurrence of mud crabs. Overall results of the study indicate a resurgence of native mud crabs at sites where sedimentary characteristics provide adequate habitat., Journal Article, Final article published