Biology Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2749
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Item In situ Activity of NAC11-7 roseobacters in Coastal Waters off the Chesapeake Bay based on ftsZ Expression(2009) Yao, Daohong; Suzuki, Marcelino T; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Phylogenetic analysis of sequences of the cell division gene ftsZ retrieved from the Atlantic coast revealed an interesting subgroup NAC11-7, which was targeted by a specifically designed and optimized Taqman assay in diel samples collected in situ and in parallel on-deck incubations. Rapid changes of ftsZ gene copies and the patchy distribution of other phylotypes at different time points suggested that different NAC11-7 populations were sampled. Strong correlations between ftsZ expression and gene abundance (r-squared=0.62), and between ftsZ expression and water temperature (r-squared=0.73) for in situ samples suggested non-synchronous growth of NAC11-7 group. Contrastingly, a sharp 9:00 AM peak of ftsZ expression in the on deck incubation experiment suggested synchronous growth. We propose a possible mixed model in which a certain fraction of the population is synchronously dividing, while a background of asynchronously dividing NAC11-7 cells also exist, some of which are expressing ftsZ at any given time.Item Genetic Determinant of Silicibacter sp. TM1040 Motility(2008-12-04) Suvanasuthi, Rooge; Belas, Robert; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Silicibacter sp. TM1040 is a member of the Roseobacter clade. Roseobacters play an important role in sulfur cycling in the ocean by degrading dimethylsulfoniopropionate (DMSP). Roseobacters are found in communities associated with most marine habits, especially with marine algae. Therefore, the ability to sense, move towards and maintain the interaction is an important physiological trait for the symbiosis between roseobacters and dinoflagellate. Previous work from our laboratory demonstrated that TM1040 is chemotaxis towards DMSP and DMSP catabolites, and motility of TM1040 is important for growth of P. piscicida. In contrast to enteric bacteria, little is known about the genes regulating motility in roseobacter species. This study, revealed similarities between the genes associated with motility in TM1040 and those from other α-proteobacteria species, but most importantly, it identified three new regulators that maybe involved in regulating the motility of TM1040.Item Mycobacteriosis in Chesapeake Bay striped bass Morone saxatilis(2008-04-22) Stine, Cynthia Bee; Kane, Andrew S.; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Striped bass, Morone saxatilis, is an economically and ecologically important species in the Chesapeake Bay and along the East coast of the United States. In 1997 an epizootic of mycobacterial infections was discovered in the Chesapeake Bay stock and subsequent reports indicated that up to three-fourths of subpopulations of striped bass in the Bay were infected, primarily older fish. This study investigated regional and age class differences in mycobacterial infections among younger striped bass in the Chesapeake Bay, and identified putative risk factors for infection. Approximately 2,000 0+ to 3+ age striped bass, a limited number of spawning stock, and bycatch species were evaluated for microbiology, histopathology and parasitology. Mycobacterial isolates were grouped according to gas chromatography fatty-acid methyl-ester profiles and multi-locus sequencing. Twenty-nine groups of mycobacteria were discerned including M. scrofulaceum, M. septicum, M. interjectum, M. triplex/M. montefiorense, M. szulgai, M. moriokaense, M. duvalii, M. avium, M. terrae, M. pseudoshottsii/M. marinum and M. shottsii, and several putative new species. The majority of mycobacteria groups observed had host overlap. Data revealed that prevalence of mycobacterial infection increased with age, up to 59%. Location of capture was associated with higher infection prevalence in fish sampled from the Pocomoke River compared with fish sampled from the Upper Bay (1+), the Choptank River (1+) and the Potomac River (0+, 1+). The presence of copepods, isopods, acanthacephalans, nematodes and trichodinid ciliates was associated with an increased prevalence odds ratio (POR) for mycobacterial infection, while the presence of bacteria other than mycobacteria was associated with a decreased POR for 0+ fish. Gender was not a risk factor for mycobacterial infection, however, gonads from some mature fish were infected. In addition, mycobacterial infections were observed in 12 other Chesapeake Bay fishes, including Atlantic menhaden, Brevoortia tyrannus, an important prey species. Mycobacterial infections in Chesapeake Bay fish appear to be more complex than the one pathogen-one host scenario. Further, vertical and food-borne transmission cannot be ruled out. Future research requires an holistic approach including evaluation of multiple host species in association with water quality and other environmental parameters.Item The role of dehalorespiring bacteria in the reductive dechlorination of polychlorinated biphenyls in Baltimore harbor sediment microcosms(2007-03-29) Fagervold, Sonja Kristine; Sowers, Kevin R; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Baltimore Harbor sediment microcosms were incubated with the 12 most predominant congeners in Aroclor 1260 and their intermediate products to identify the major dechlorination pathways. Most congeners were dechlorinated in the meta position, although some dechlorination in the para and ortho positions was observed. The major dechlorination products were tetrachlorinated biphenyls with unflanked chlorines. Specific dechlorination rates of parent and intermediate PCB congeners were determined to identify the rate limiting reactions. To identify the microorganisms responsible for the dechlorination pathways, I developed PCR primers specific for the 16S rRNA genes of known PCB dehalogenating bacteria. These PCR primers were used in conjunction with DGGE to selectively identify the microorganisms that catalyzed each dechlorination reaction. Only three phylotypes were identified that catalyze the dechlorination of Aroclor 1260, and the selective activities of these phylotypes were determined. Phylotype DEH10 had high sequence similarity to Dehalococcoides spp., while phylotype SF1 had high sequence similarity to the o-17/DF-1 group of PCB dechlorinating bacteria. The third phylotype had 100% sequence similarity to the ortho-dechlorinating bacterium o-17 described previously from Baltimore Harbor sediments. Most dechlorination reactions for all three phylotypes were growth-linked, indicating that PCB-impacted environments have the potential to sustain populations of PCB dechlorinating organisms. To investigate whether bioaugmentation would be feasible for bioremediation of PCB contaminated sites, Baltimore Harbor sediment microcosms were supplemented with known dechlorinators and their effects on PCBs dechlorination patterns determined. The addition of different dechlorinators resulted in different dechlorination patterns. Finally, novel putative reductive dehalogenases were identified from the PCB dechlorinating bacterium DF-1 using degenerate PCR primers. Comparative sequence analyses indicated that they had high sequence similarity to both confirmed and putative dehalogenases from several Dehalococcoides species. In conclusion, microorganisms that can dechlorinate Aroclor 1260 have been identified for the first time and dechlorination of congener mixtures was shown to occur by the growth-linked complementary activities of bacterial consortia within the Chloroflexi. Demonstration that bioaugmentation with these organisms can influence rates and pathways of dechlorination, combined with the development of molecular assay for monitoring their fate, provide potentially valuable tools for the development of bioremedial strategies for PCB contaminated sediments.Item Ecological Significance of Luminescence in Vibrio cholerae: Occurrence, Structure, Expression, and Function(2006-11-25) Grim, Christopher John; Colwell, Rita R; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Several Vibrio species are bioluminescent, including Vibrio cholerae. Analysis of 224 non-pathogenic V. cholerae isolates collected from the Chesapeake Bay, MD, revealed that 52% were luminescent, and 58% of the isolates harbor the luxA gene. A significant association of luxA to the gene stn (r = 0.40) was observed and luminescent strains were found to have a significant association with sample fraction and time of sampling, especially from the observed interaction of these two traits. In contrast, 334 non-pathogenic V. cholerae strains isolated from two rural provinces in Bangladesh, yielded 21 luminescent (6.3%) and 35 luxA+ (10.5%) isolates. None (0%) of 48 laboratory reference pathogenic strains from various geographic locations or 222 environmental and clinical isolated strains of V. cholerae O1 or O139 from Bangladesh were luminescent or harbored the lux operon. To improve success of isolation of V. cholerae from environmental samples, two colony blot hybridization methods were developed. Specificity of two probes was confirmed, using laboratory reference strains, in addition to environmental and clinical isolates, and sensitivity of the probes was confirmed using water samples into which V. cholerae had been inoculated. The lux operon of V. cholerae was sequenced and its chromosomal location determined. The operon organization is most similar to that of Shewanella hanedai and the non-luxF Photobacterium leiognathi. Sequence analysis revealed that the V. cholerae lux operon is most similar in its genomic sequence to V. harveyi and Photorhadbus luminescens and it most likely originated from a common Vibrionaceae ancestor. Using a new bioassay method that measures expression level, several classes of defective luminescent V. cholerae were identified and characterized, including one class previously termed dark, or K variants, in V. harveyi. Multiple causes of the defects were identified, indicating several levels of luminescence control in V. cholerae, in addition to autoinduction and lux repression. Using luxA mutants, luminescence was implicated in conveying competitive advantage in growth under microaerophilic conditions, DNA repair by photoreactivation, and neutralization of reactive oxidative species. These results demonstrate that bioluminescence is a frequently occurring trait in non-pathogenic V. cholerae, the expression of which gives a selective advantage in specific habitats.Item Isolation and characterization of a sponge-associated actinomycete that produces manzamines(2006-11-20) Peraud, Olivier; Hill, Russell T; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Two Indonesian sponges, Acanthostrongylophora sp. Sponge 35 and Sponge 52, containing manzamine A were collected off the coast of Manado, Indonesia. Manzamines are a family of marine alkaloids that exhibit a complex molecular architecture and possess bioactivities including antitumour, antimicrobial, antiparasitic and insecticidal activities. Manzamines have been found in 17 different species of sponges with wide geographical distribution which has led to speculation that they may be produced by a microbial symbiont rather than by the sponges themselves. The sponges' microbial communities were investigated using 16S rRNA gene analysis and a rational culture-based microbiology approach in which specific bacterial groups were targeted. The molecular analysis of these microbial communities revealed that they were complex and diverse. Microbiological analyses were conducted on Acanthostrongylophora sp. with a particular emphasis on the isolation of actinomycetes because of the high number of actinomycete sequences in this sponge 16S rRNA gene clone library and their excellent track record as bioactive compound producers. One of the isolated actinomycetes, Micromonospora sp. strain M42, produces manzamine A and 8-hydroxy-manzamine, compounds initially detected in the sponge. A detailed analysis of Micromonospora sp. strain M42 showed that it grew on a wide range of salt concentrations with an optimal growth at 0-1% NaCl. Cultures of Micromonospora sp. strain M42 consistently produced manzamine A with a maximum yield of 1 mg/l. The genome size of Micromonospora sp. strain M42 was estimated at 6.7 Mb by pulsed field gel electrophoresis. The biosynthetic gene pathway encoding manzamine A was investigated using both biochemistry and molecular methods yet it remains elusive. Micromonospora sp. strain M42 underwent UV mutagenesis leading to isolation of mutants with yield of manzamine A improved by 3.5 fold. One of the mutants produces manzamine B, the putative biosynthetic precursor of manzamine A. A fosmid library of Micromonospora sp. strain M42 was constructed and low-pass genome sequencing gave insights into the strain's genome and revealed a high number of genes devoted to the production of secondary metabolites including polyketides and non-ribosomal peptides. The isolation of Micromonospora sp. strain M42 greatly improves the chances of manzamines becoming a drug class for treatment of malaria.Item Comparative genomic analysis of Vibrio cholerae O31: capsule, O-antigen, pathogenesis and genome(2006-11-21) Chen, Yuansha; Morris, J Glenn; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Vibrio cholerae is the causative agent of cholera. In order to understand the genetic basis underlying the emergence of novel epidemic strains of V. cholerae, the genetics of surface polysaccharide biogenesis, and the role of lateral gene transfer in the evolution of this species, we investigated. NRT36S and A5 are both NAG-ST producing, cholera toxin negative, serogroup O31 V. cholerae. NRT36S is encapsulated and causes diarrhea when administered to volunteers; A5 is acapsular and does not colonize or cause illness in humans. The structure of the capsular (CPS) polysaccharide in NRT36S was determined by NMR. The gene cluster of CPS biogenesis was identified by transposon mutagenesis combined with whole genome sequencing data. The CPS gene cluster shared the same genetic locus as that of the O-antigen of lipopolysaccharide (LPS) biogenesis gene cluster. The LPS biogenesis regions in A5 were similar to NRT36S except that a 6.5 kb fragment in A5 replaced a 10 kb fragment in NRT36S in the middle of the LPS gene cluster. The genome of NRT36S was sequenced to a draft containing 174 contigs plus the superintegron region. Besides confirming the existence of NAG-ST, we also identified the genes for a type three secretion system (TTSS), a putative exotoxin, and two different RTX genes. Four pili systems were also identified. Therefore, the genome of non-O1 Vibrio cholerae NRT36S demonstrates the presence of pathogenic mechanisms that are distinct from O1 V. cholerae. We conclude that lateral gene transfer plays a critical role in the emergence of new strains. The co-location of CPS and LPS could provide a mechanism for simultaneous emergence of new O and K antigens in a single strain. Our data also highlights the apparent mobility within the CPS/LPS region that would provide a basis for the large number of observed V. cholerae serogroups and the emergence of novel epidemic strains.Item Bacterioplankton in the Chesapeake Bay: Genetic diversity, population dynamics and community proteomics(2006-10-10) Kan, Jinjun; Chen, Feng; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Although the ecosystem of the Chesapeake Bay has been studied extensively, little is known about the genetic diversity, population dynamics and metabolic activity of bacterioplankton living in the Bay. In this study, clone libraries containing the rRNA operon (16S rRNA-ITS-23S rRNA) were constructed from samples collected from the Chesapeake Bay to study spatial and temporal dynamics of estuarine bacterioplankton. Major bacterial groups changed dramatically between cold and warm seasons. In the summer, Alpha- and Gammaproteobacteria, Bacteroidetes (Flavobacterium-Bacteroidetes-Cytophaga), Cyanobacteria and Actinobacteria were the dominant groups while in the winter, Alpha- and Betaproteobacteria, and Actinobacteria were commonly found. Clone library analysis also revealed dramatic shifts in bacterial species composition between seasons. Unique SAR11, SAR86, and Roseobacter clades were discovered in the Chesapeake Bay, suggesting the ecological adaptation of organisms endemic to the Bay or perhaps, large temperate estuaries. The bacterioplankton populations were monitored from 2002 to 2004 by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments. Remarkable seasonal shifts and repeatable annual patterns were identified. Temporal variation of bacterial communities was best explained by the change of chlorophyll a (Chl a) and water temperature, while other factors such as dissolved oxygen, ammonia, nitrite and nitrate, and viral abundance also contributed to the seasonal succession of bacterial populations. In order to understand ecological functions of microbes living in the natural environment, a community-based proteomic approach was developed. Typically, a few hundred-protein spots were visualized based on two-dimensional gel electrophoresis (2-DGE) from Chesapeake Bay microbial communities (0.2 to 3.0 µm filtered fractions). Distinct seasonal patterns and noticeable spatial variations of Chesapeake Bay metaproteomes were observed and the metaproteomic patterns correlated with genetic fingerprints based on 16S rRNA-DGGE. Six protein spots were characterized by LC-MS/MS and three of them were most closely related to the genes in the Sargasso Sea metagenomic database. We proved for the first time that metaproteomics could be applied to a complex marine microbial community. Our results indicate that community proteomics has great potential to unveil novel microgeochemical functions and to link microbial functions to their population structures.Item A Theoretical Microbial Contamination Model for a Human Mars Mission(2006-01-31) Lupisella, Mark Lewis; Sebens, Kenneth P.; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Contamination from a human presence on Mars could significantly compromise the search for extraterrestrial life. In particular, the difficulties in controlling microbial contamination, the potential for terrestrial microbes to grow, evolve, compete, and modify the Martian environment, and the likely microbial nature of putative Martian life, make microbial contamination worthy of focus as we begin to plan for a human mission to Mars. This dissertation describes a relatively simple theoretical model that can be used to explore how microbial contamination from a human Mars mission might survive and grow in the Martian soil environment surrounding a habitat. A user interface has been developed to allow a general practitioner to choose values and functions for almost all parameters ranging from the number of astronauts to the half-saturation constants for microbial growth. Systematic deviations from a baseline set of parameter values are explored as potential plausible scenarios for the first human Mars missions. The total viable population and population density are the primary state variables of interest, but other variables such as the total number of births and total dead and viable microbes are also tracked. The general approach was to find the most plausible parameter value combinations that produced a population density of 1 microbe/cm3 or greater, a threshold that was used to categorize the more noteworthy populations for subsequent analysis. Preliminary assessments indicate that terrestrial microbial contamination resulting from leakage from a limited human mission (perhaps lasting up to 5 months) will not likely become a problematic population in the near-term as long as reasonable contamination control measures are implemented (for example, a habitat leak rate no greater than 1 % per hour). However, there appear to be plausible, albeit unlikely, scenarios that could cause problematic populations, depending in part on (a) the initial survival fraction and death rate of microbes that are leaked into the Martian environment, which depends largely on the possibility for protection from the high UV radiation environment on Mars, (b) organic nutrient availability, and (c) liquid water availability, which is likely to be the limiting survival and growth factor.Item Expression of recombinant proteins in the methane-producing archaeon Methanosarcina acetivorans(2005-12-16) MacAuley, Sheridan Rose; Sowers, Kevin R; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Recombinant protein expression is a necessary tool for the investigation of proteins in the post-genomic era. While many systems exist for recombinant protein expression in organisms of the eukaryotic and eubacterial domains, few to none are available in the Archaea. A recombinant protein expression system using the methanogenic archaeon Methanosarcina acetivorans was developed which uses the highly regulated cdh promoter and allows expression of recombinant protein with optional 6xHis protein fusions to facilitate rapid purification. A protocol for high-density mass cultivation of M. acetivorans in a stainless steel bioreactor configured as a pH-auxostat was developed. The cdh promoter and alternate promoters were analyzed in attempt to enhance expression of recombinant proteins. The protein expression system was tested on several proteins: the Methanocaldococcus jannaschii prolyl tRNA synthetase, the M. acetivorans prolyl tRNA synthetase, the Methanosarcina thermophila carbonic anhydrase, and the Dehalococcoides ethenogenes tricholorethylene dehalogenase.