Poster Abstracts Marine Biomedicine and Environmental Sciences Research Open House

27 July 2001

(Student names in blue)

 

Characterization of a Renal Urea Transporter from a Euryhaline 

Elasmobranch, Dasyatis sabina.

Michael G. Janech1 , Ruihua Chen2 , Richard V Paul2 , Mark W Nowak3  Donald H. Miller3 , David W Ploth2,4 , Wayne R Fitzgibbon2 : 1Medical University of South Carolina - Marine Biomedicine and Environmental Sciences, 221 Fort Johnson Rd., Charleston, South Carolina 29412, 2Nephrology, 96 Jonathan Lucas, Charleston, SC 29425, 3Pharmacology, 171 Ashley Ave., Charleston, SC 29425; 4VAMC, Charleston, SC

Regulation of tubular urea reabsorption appears to be an important mechanism in the regulation of fluid homeostasis by elasmobranch fishes (sharks, skates, and rays). Urea movement across renal tubular epithelia is primarily mediated by a family of specialized, facilitated transport proteins. Recently, we have identified and sequenced a 2.6kb cDNA in the kidney of the euryhaline stingray, Dasyatis sabina. This cDNA encodes for a 431 amino acid protein that is homologous to the previously reported shark urea transporter. The aim of this study was to verify that the transcript encoded a functional stingray urea transporter (STRUT). The uptake of [14C]urea by Xenopus oocytes injected with 50ng STRUT cRNA was used to determine urea transporter function. Water-injected oocytes served as controls. In STRUT cRNA-injected oocytes [14C]urea uptake was elevated 12-fold above water-injected controls (mean ± SEM; 55 ± 7 vs. 5 ± 1 pmol oocyte-1 90s-1 , respectively). Preincubation of STRUT-injected oocytes with 0.5mM phloretin for 30 min. inhibited [14C]urea uptake by 80% (11 ± 2 pmol oocyte-1 90s-1 ). We tested whether STRUT-mediated uptake of urea was dependent on the presence of NaCl in the bathing media by replacing NaCl with 200mM mannitol. The absence of NaCl did not alter STRUT-mediated urea uptake by oocytes (NaCl: 55 ± 7 vs. mannitol: 57 ± 11 pmol oocyte-1 90s-1 ). Further, preincubation with 150mM urea, acetamide, or trimethylamine oxide did not result in a significant reduction in urea uptake, whereas, thiourea, methylurea, and 1,3 dimethylurea resulted in a significant reduction in urea uptake. These data suggest that STRUT is a phloretin-sensitive urea transport protein similar in sequence and function to the UT-A family of urea transporters.

 

Pseudomonas aeruginosa Cell Surface Adaptation During Growth on Crude Oil

 

R. Sean Norman 1 and Pamela J. Morris1,2

1 Marine Biomedicine and Environmental Sciences

2 Microbiology and Immunology, Medical University of South Carolina

  To evaluate microbial cell surface adaptation to growth on crude oil, we used Pseudomonas aeruginosa strains isolated from a crude oil-degrading enrichment culture.  Strain U1 forms large, smooth, slightly mucoid colonies when grown on LB agar while P. aeruginosa U3 forms small, rough, dry colonies.  U1 is also characterized by an extended lag when Bonny Light crude oil (BLC) is used as the sole carbon source while U3 demonstrates reduced lag when grown on BLC.  Cultures were established using a soluble carbon source (glucose) or an insoluble carbon source (Bonny Light crude oil, BLC).  Cultures were also established on glucose or BLC and supplemented with EDTA, a cell surface destabilizing reagent.  Cultures on BLC were sampled every 8-16 hrs for 200 hrs, while cultures on glucose were sampled every 4-24 hours for 64 hours.  At each time point, one set of triplicate samples was used to determine bacterial growth and LPS cell surface release.  Also, at each time point, a second set of triplicate samples was analyzed by gas chromatography to determine n-alkane degradation.  Results demonstrate that both U1 and U3 are capable of growth on BLC reaching cell densities of 1.9E+09 cells•ml-1 by 128 hrs and 1.0E+09 cells•ml-1 by 80 hrs, respectively.  However, U1 and U3 on BLC and amended with EDTA demonstrate reduced growth with cell densities reaching 6.6E+07 by 128 hrs and 6.4E+07 by 80 hrs, respectively.  For comparison, cultures grown on glucose demonstrated similar growth rates with and without EDTA amendment.  LPS release into the culture supernatant was estimated by measuring the quantity of 2-keto-3-deoxyoctonic acid (a core LPS sugar).  These data demonstrate a maximum release of LPS by the U1 and U3 BLC cultures of 0.09µg·µl-1 by 104 hrs and 0.17µg·µl-1 by 40 hrs, respectively.  Percent n-alkane degradation by U1and U3 paralleled growth and LPS release resulting in 100% degradation by 104 and 40 hrs, respectively.  These results demonstrate that P. aeruginosa adapts to growth on crude oil by releasing cell surface LPS molecules, increasing the cell surface hydrophobicity.  At present, the surface morphology and hydrophobicity of cells grown on BLC with and without EDTA are being examined by atomic force microscopy.     

 

Diversity of the Penaeidin Antimicrobial Peptide Family in Two Species of Shrimp

Brandon J. Cuthbertson1,3, Eleanor F. Shepard2,3, Robert W. Chapman2,3, Gregory W. Warr1,3,

and Paul S. Gross1,3

 

1Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC,   2Marine Resources Research Institute, South Carolina Department of Natural Resources, Charleston, SC ,  3Marine Biomedicine and Environmental Sciences Program, Medical University of South Carolina, Charleston, SC

 Penaeidins, a unique family of antimicrobial peptides with both proline and cysteine-rich domains, were initially identified and characterized as peptides in the hemolymph of the Pacific white shrimp, Litopenaeus vannamei. Subsequently, cDNA cloning from a library made from pooled hemocytes permitted the identification of 3 classes of penaeidin. We report here the results of an investigation of penaeidin diversity in two species of shrimp, L. vannamei and L. setiferus (Atlantic white shrimp). This study revealed that (1) a novel penaeidin class, designated penaeidin 4, is present in both L. vannamei and L. setiferus, (2) all classes of penaeidins are expressed in an individual shrimp, (3) penaeidin sequence variability (especially of class 3), within and between individuals, is not interpretable as simple allelic polymorphism, (4) penaeidins are encoded by a small gene family or multi-component locus,and (5) based on phylogenetic analysis, penaeidins fall into three classes.  In this analysis classes 1 and 2 are combined, with classes 3 and 4 remaining distinct.  All classes of penaeidin were present prior to the speciation of L. vannamei and L. setiferus.

 

The Cloning and Characterization of Metallothionein Isoforms from

Crassostrea virginica 

Matthew J. Jenny1,  Amy H. Ringwood1,2, Eric R. Lacy1,3 Robert W. Chapman1,2, and  Gregory W. Warr1,4

1Marine Biomedicine and Environmental Studies, MUSC, 2Marine Resources Research Institute, SC Department of Natural Resources, 3Department of Cell Biology and Anatomy, MUSC, and 4Department of Biochemistry and Molecular Biology, MUSC

 The American oyster, Crassostrea virginica, is an important indicator species of estuarine health and a valuable model for evaluating the relationship between ecosystems and human health.  Metallothioneins are low-molecular-weight proteins with characteristic repeating cysteine motifs.  They function in metal metabolism, homeostasis and detoxification.  To date only one protein has been identified in C. virginica. To further understand the role of metallothioneins in oysters, cDNA libraries were constructed from control, Cd-, and Cu-treated C. virginica embryos.  Random probes were generated from a vector containing the Cd-MT of C. virginica (provided by Roesijadi, G.) and used to probe the respective libraries.  Preliminary sequencing has identified multiple MT isoforms from all three libraries.  In addition to the expected ~7kDa protein, we have identified nucleotide sequences coding for ~4-5 kDa as well as ~15kDa proteins.  Unique to the Cd-treated library are cDNAs representing splice variants of the original oyster MT which code primarily for the b-domain.  This suggests the formation of one-domain MT proteins that may be energetically favorable detoxification pathways. Random EST screenings have identified a novel MT whose primary structure is ~40% homologous to the original oyster MT.  These results likely represent the presence of two distinct MT gene families in oysters. 

 

The Impact of pH on Microbial Ni Toxicity

J. D. Van Nostrand1, T. V. Khijniak1, A. Sowder2, P. M. Bertsch2 and P. J. Morris1

1Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC, 2Savannah River Ecology Laboratory, The University of Georgia, Aiken, SC, 3Marine Biomedicine and Environmental Sciences, Medical University of South Carolina, Charleston, SC

 The U.S. Department of Energy's Savannah River Site was a nuclear materials processing facility that discharged metallurgical process wastes that led to extensive co-contamination of groundwater, stream sediments, and soil with chlorinated solvents and heavy metals.  Approximately 42,000 kg of uranium (U) and similar concentrations of nickel (Ni) are present, ranging from 2 to 5300 ppm Ni and <1 to 6100 ppm U in soils and sediments.  Ni appears to be providing a greater selection pressure on the microbial community than U, with more Ni (maximum 100 ppm) found in the porewater (where it would be accessible to the microorganisms) than U (maximum 10 ppm).  Four Gram-positive organisms (Arthrobacter oxydans, Streptomyces galbus, Streptomyces aureofaciens, and Kitasatispora cystarginea) were isolated in the presence of 5000 ppm Ni and identified using 16S rDNA sequence analysis.  Studies with S. aureofaciens suggested that Ni was less toxic at pH 5 than pH 6 or 7.  This suggests that either Ni speciation is altered by pH to a less toxic/bioavailable form, or that Ni resistance mechanisms are more efficient during growth at lower pH.  Since growth of S. aureofaciens is not substantial at pH 5, we chose to study Ni toxicity in Burkholderia cepacia PR1, a constitutive TCE degrader, which grows well at pH 5, 6, and 7.  B. cepacia PR1 was able to grow on higher Ni concentrations at pH 5 (1000 ppm) compared to pH 6 (200 ppm) and 7 (50 ppm).  Studies using the MinteqA2 model to determine Ni speciation using our media components indicate that there is no speciation difference at pH 5, 6, or 7 at 50 or 100 ppm Ni.  At 1000 ppm, the Ni begins to precipitate at pH 7, causing the Ni to be less available and potentially less toxic.  However, the speciation results and our experimental data do not correlate, since Ni toxicity was greatest at pH 7.  Our on-going studies will address the hypothesis that active Ni efflux functions more efficiently in B. cepacia PR1 at lower pH values due to higher environmental H+ concentrations.

Joy Van Nostrand is  Ph.D. student in the Department of Microbiology & Immunology.  She conducts her research on the Ft. Johnson campus.

 

 

Functional Analysis of the Eµ3’ Enhancer of the Catfish

 Christopher C. Cioffi 1, Darlene L. Middleton 2, Melanie R. Wilson 3, Norman W. Miller 3, L. William Clem 3, and Gregory W. Warr 2,4

 1Departments of Microbiology and Immunology, and  2Department of Biochemistry and Molecular Biology, The Medical University of South Carolina, Charleston, SC, 3Department of Microbiology, University of Mississippi Medical Center, Jackson MS,  and  4Marine Biomedicine and Environmental Sciences, Medical University of South Carolina, Charleston, SC 

The transcriptional enhancer of the IgH locus of the channel catfish, Ictalurus punctatus, shows strong B-cell specific activity. This transcriptional enhancer (Eµ3’) differs from the mammalian Eµ enhancer in both location and structure.  Eµ3’, which occurs between the m and d genes, contains numerous transcription factor binding sites, predominantly octamer and µE5 motifs of both consensus and variant sequences.  It lacks the classical µA-TFE3(CBF)-µB core array of binding motifs seen within mammalian IgH Eµ enhancers.  To determine the functional DNA binding motifs required for activity within this enhancer, a series of mutant enhancers was created.  Specific DNA binding motifs were mutated within the native Eµ3’ enhancer, using sequence-targeted PCR. While the mutation of consensus and variant octamer motifs, (individually or in multiples) decreased enhancer function, mutation of a single consensus mE5 motif had the most profound effect, and was sufficient to destroy the function of this enhancer as tested in mammalian plasmacytomas. Mutation of this consensus mE5 site, combined with mutations of certain octamer sites, was sufficient to destroy function in catfish B cells.  Experiments using artificial enhancers containing multimers of motifs or short regions of the native enhancer suggested that the minimal Em3’ enhancer a) contains a consensus mE5 site and 2 octamer sites, b) is B cell specific, and c) is active across species.  The dependence of an Ig enhancer on sites that bind bHLH and Oct transcription factors has not previously been observed, and confirms large differences in structure and function between fish and mammalian IgH enhancers.

*This work was supported by awards from the National Science Foundation (MCB9722996 and MCB9807531) and the NIH (R37-AI-19530).

 

Impact of Anaerobic Dechlorination on PCB Toxicity

            Ashley Smithwick1, John Quensen2, Andrew Smith3, Lucille London1,3, and Pamela J. Morris1,3

 

  1Marine Biomedicine and Environmental Sciences, 2Crop and Soil Sciences, Michigan State University, East Lansing, Michigan 48824, 3Microbiology and Immunology, Medical University of South Carolina, Charleston, SC 29403.

Polychlorinated biphenyls (PCBs) are widespread environmental contaminants that were used over a period of fifty years for such industrial applications as dielectric fluids and flame retardants.  As a result of the lipophilic nature of the compounds, PCBs bioaccumulate in organisms and persist in soil and sediments.  Upon entering the environment, PCBs can be degraded by many processes, including microbial reductive dechlorination in which chlorines are removed preferentially from the meta- and para- positions of the biphenyl ring resulting in a PCB mixture that is less chlorinated and enriched in ortho- substituted congeners.  In this study, Aroclor 1242 was anaerobically dechlorinated using inoculum from three PCB-contaminated sites, the Hudson River (New York), Silver Lake (Massachusetts), and River Raisin (Michigan).  The immunotoxicity of these mixtures was examined using lymphocytes isolated from the spleens of C57 mice, which express a high affinity Ah receptor.  Immunotoxicity was assessed by lymphocyte viability, LPS-induced splenocyte proliferation, and antibody secretion following PCB exposure.  No change in viability was observed between Aroclor 1242 and dechlorinated Aroclor mixtures as measured by trypan blue exclusion.  A 30% decrease in LPS-induced splenocyte proliferation was observed following exposure to Aroclor 1242 and similar inhibition was observed following exposure to three dechlorinated Aroclor 1242 mixtures. Secreted antibody concentrations were also determined following exposure of lymphocytes to PCB mixtures using an enzyme-linked immunosorbent assay (ELISA).  An inhibition of antibody secretion was noted at similar concentrations of Aroclor 1242 and two dechlorinated samples, River Raisin and Silver Lake; however, secreted antibodies were not detected following exposure of lymphocytes to the dechlorinated Aroclor 1242 mixture, Hudson River, suggesting this dechlorinated mixture may be more toxic than the parent Aroclor.

 

Mechanisms and Microbial Community Structure of C30 17a,21b(H)-Hopane Transformation by a Crude Oil-Degrading Enrichment Culture

Dan Bost1, Jeff Retzke2, Amanda Graham3, Brenda Ross4, Peter Moeller5,6, and Pam Morris1,6

1Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC, 2Masters in Environmental Sciences Program, University of Charleston, Charleston, SC,   3Molecular &  Cellular Biology and Pathobiology, Medical University of South Carolina, Charleston, SC,  4Department of Chemistry, Cottey College, Neveda, MO, 5Center for Coastal Environmental Health & Biomolecular Research, National Ocean Service, Charleston, SC,  6Marine Biomedicine and Environmental Sciences, Medical University of South Carolina, Charleston, SC

The potential mechanisms of transformation of the C30 17a,21b(H)-hopane petroleum biomarker were examined using a microbial enrichment culture (HEFDLC) developed using a C30 17a,21b(H)-hopane-enriched fraction (HEF) of Bonny Light crude oil.  Heterotrophic plate counts of HEFDLC on HEF after one month at 30oC and shaking at 200 rpm revealed an order of magnitude increase in cell number, reaching stationary phase at seven days.  DGGE analysis of HEFDLC revealed no dramatic community profile differences over the course of the incubation.  Analysis of culture extracts using gas chromatography with flame ionization detection revealed hopane degradation relative to the conserved C30 18a(H)-oleanane, as indicated by a 196% increase in the oleanane to hopane ratio.  However, given this difference in susceptibility of hopane and oleanane to microbial transformation, the pentacyclic structures of these molecules differ only in their E ring moieties, a 1,1-dimethylcyclohexane (DMC) for C30 18a(H)-oleanane and an isopropylcyclopentane (ICP) group for C30 17a,21b(H)-hopane.  HEFDLC was transferred (4%) into a minimal medium containing 500 ppm of either ICP or DMC and placed at 30oC for one month.  On the ICP moiety, heterotrophic plate counts increased two orders of magnitude by day seven, while counts of HEFDLC on DMC were comparable to those on the minimal medium alone.  Growth was not observed on the corresponding unsubstituted cycloalkanes, cyclohexane and cyclopentane.  These data suggest that the initial attack on C30 17a,21b(H)-hopane occurs on the alkyl side chain of the E ring moiety.  Previous studies in the laboratory have demonstrated the concomitant aerobic degradation of hopanes and 25-norhopanes, the putative demethylation products of the hopanes, suggesting that both compounds share a common mechanism of degradation.  The present data support a possible mechanism of initial transformation for complex saturated biomarkers with similar alkyl groups.

 

 

Macromolecular Responses to Atrazine Exposure in a Suite of Phytoplankton Species

 

J.A. Weiner1, M.E. DeLorenzo2, and M.H. Fulton2

 

1Marine Biomedicine and Environmental Sciences, Medical University of South Carolina, Charleston, SC 2Center for Coastal Environmental Health & Biomolecular Research, National Ocean Service,  Charleston, SC

Contaminant-induced macromolecular changes of phytoplankton may compromise the nutritional status of grazers.  This study quantified macromolecular changes of Dunaliella tertiolecta, Selenastrum capricornutum, Phaeodactyla tricornutum, Isochrysis galbana, and Synechococcus sp. in response to atrazine.  Growth rate 96-hour EC50 values for these species ranged from 37.20 mg/L to 104.11 mg/L.  Algae were exposed to three atrazine treatments: 0.5x EC50, 1x EC50, and 2x EC50 for 96 hours.  Following exposure, a fractionation assay was used to quantify total 14C uptake and percent of 14C assimilated into low molecular weight (LMW) molecules, lipids, polysaccharides, and protein.  Despite decreased growth rate in all species, total carbon uptake responses varied.  Total carbon uptake was unaffected in S. capricornutum, I. galbana, and Synechococcus sp., while D. tertiolecta and P. tricornutum showed decreased uptake with atrazine exposure.  In S. capricornutum, percent of LMW molecules increased and percent protein per cell had a decreasing trend with increasing atrazine concentrations.  D. tertiolecta showed an increase in percent LMW macromolecules and a decrease in percent protein per cell with atrazine exposure (p £ 0.05).  D. tertiolecta also had increased percent of lipid and polysaccharide per cell at the highest atrazine treatment (p £ 0.05).  Synechococcus sp. showed a decrease in percent of LMW molecules and an increase in percent protein per cell with atrazine exposure (p £ 0.05).  No significant changes were found in P. tertiolecta or I. galbana exposed to atrazine, suggesting class-specific differences in phytoplankton response to atrazine.  These data indicate a common chlorophyte response of increasing percent of LMW molecules and decreasing percent of protein per cell with atrazine exposure.  Atrazine exposure also alters in molecular composition of Synechococcus sp., but does not effect P. tricornutum or I. galbana.  These alterations may alter the nutritional value of microalgae.  Future research will evaluate nutritional effects on higher trophic levels.

 

Isolation  and Characterization of a Putative 11.5 kDa Antimicrobial Protein from the Penaeid Shrimps, Litopenaeus vannamei and Litopenaeus setiferus

Thomas C. Bartlett1, Paul S. Gross2,4, Robert W. Chapman3,4, Brandon J. Cuthbertson2,4, Eleanor F. Shepard3,4, and Gregory W. Warr2,4

1Marine Biology Masters Program, University of Charleston, Charleston, SC, 2Department of Biochemistry and Molecular Biology, MUSC, Charleston, SC, 3Marine Resources Research Institute, SC Department of Natural Resources, Charleston, SC, 4Marine Biomedicine and Environmental Sciences Program, MUSC, Charleston, SC

          The Pacific white shrimp, Litopenaeus vannamei, and the Atlantic white shrimp, L. setiferus, are two commercially important species in the Penaeid family. L. vannamei is cultured circumtropically, while L. setiferus is a mainstay of trawl fisheries on the US mid-Atlantic coast. The effects of pathogens can devastate shrimp populations, particularly cultured L. vannamei where high-density aquaculture allow rapid spread of infection. Crustacean response to pathogens, although poorly understood, is believed to rest entirely with innate, non-adaptive mechanisms including phagocytosis, encapsulation, clotting, and antimicrobial activities.  In an attempt to better understand the immune system in penaeid shrimp, a survey of the expressed genes was performed using an Expressed Sequence Tag (EST) approach. From the 1049 ESTs isolated from the hemocytes of both L. vannamei (504 ESTs) and L. setiferus (545 ESTs), 21 (14 from L. vannamei and 7 from L. setiferus) demonstrated approximately 40% amino acid sequence homology with an11.5 kD antimicrobial protein previously found in the shore crab, Carcinus maenas. Initial sequencing of the ESTs shows the presence of at least five different gene isoforms, two in L. vannamei and three in L. setiferus. Analysis of a non-PCR based L. vannamei genomic library reveals the presence of three possible isoforms that possess amino acid substitutions different from those seen in the L. vannamei EST library. In addition, genomic southern blotting reveals a complex banding pattern which indicates that the homolog may be encoded by one (or possible two) genes that display allelic polymorphism. In order to characterize the antimicrobial activity of the message, full length and truncated clones will be expressed in a pQE-TriSystem vector and the protein products used in antibacterial assays.

 

Functional Differences in Transport Capability of Pgp in Eastern Oysters (Crassonstrea virginica) from a Clean and a Polluted Site

T.J. Christl1, Karl Karnaky Jr.2, Geoff Scott3­

1 Medical University of South Carolina, Marine Biomedicine and Environmental Sciences, Charleston, SC;  2  Medical University of South Carolina, Department of Cellular Biology and Anatomy, Charleston, SC; 3  National Ocean Service, Center for Coastal and Environmental Health and Biomolecular Research, Charleston, SC

        Expression of drug transport proteins such as P-glycoprotein (Pgp) may help reduce negative effects related to exposure of marine animals to chemical pollutants.  Modulation of these proteins can help provide protection against pollutants when necessary, and conserve energy when the animal is not being challenged by contaminants.  Gill tissue from oysters collected from a polluted site (Koppers Creek, Charleston, SC) and an historically clean site (Leadenwah Creek, Charleston, SC) were compared for their ability to transport a model Pgp substrate, Rhodamine 123, and the ability of that transport to be inhibited by a model Pgp inhibitor, Verapamil.  Inhibition of Rhodamine 123 transport by verapamil ranged from -5.4% to 36.5% in gill tissue of oysters taken from Koppers Creek.  Staining by Rhodamine 123 was seen homogeneously throughout the tissue of Koppers Creek oysters.  In contrast, gill tissue from Leadenwah Creek oysters displayed a heterogeneous pattern of staining by Rhodamine 123, with no apparent pattern based on cell type.  Transport function of oyster gills tissue from Leadenwah Creek are ongoing and will be included with the poster presentation and will be discussed.