Nadathur S. Govind

SMALL‐SUBUNIT RIBOSOMAL DNA SEQUENCE ANALYSES AND A RECONSTRUCTION OF THE INFERRED PHYLOGENY AMONG SYMBIOTIC DINOFLAGELLATES (PYRROPHYTA)1

The small‐subunit ribosomal RNA genes (SSU rDNA) from the four symbiotic dinoflagellates, Symbiodinium corculorum Trench isolated from the bivalve mollusc Corculum cardissa (from Belau, Western Caroline Is.), S. meandrinae Trench, from the scleractinian coral Meandrina meandrites (from famaica, W.I.), Gloeodinium viscum Banaszak et al. from the hydrocoral Millepora dichotoma (from the Gulf of Aqaba), and Amphidinium belauense Trench from the acoel flatworm Haplodiscus sp. (from Belau) have been amplified by the polymerase chain reaction, cloned, and sequenced. Following alignment of these complete sequences to homologous sequences from six other dinoflagellates, eight api‐complexans, six ciliates, six chromophytes and oomycetes, three ascomycetes, two rhodophytes, two chlorophytes, and two myxomycetes (with Physarum polycephalum as the outgroup), phylogenetic reconstruction was conducted using Fitch and Margoliash distance, DNA maximum likelihood, and Wagner parsimony methods, with bootstrap resampling. All methods generated trees with similar topologies. The inferred “across Kingdom” phylogeny reemphasizes previous reports that show that the dinoflagellates, the apicomplexans, and the ciliates share a common ancestry and that the dinoflagellates are distantly related to the chromophyte‐oömycete lineage. The evidence supports the concept of a polyphyletic origin of dinoflagellate‐invertebrate symbioses, as symbiotic dinoflagellates represent seven genera in at least four orders. The three symbiotic species, S. corculorum, S. meandrinae, and S. pilosum, consistent with their morphological and biochemical similarities, cluster most closely. Symbiodinium pulchrorum Trench, the symbiontfrom the Hawaiian sea anemone Aiptasia pulchella, is more distantly related to them. Gloeodinium viscum is not closely related to the Symbiodinium species. Amphidinium carterae (free‐living) and A. belauense (symbiotic) also appear to be distantly related to Symbiodinium. Some symbionts (e.g. S. corculorum, S. pilosum) from distant geographic locations (the Indo‐Pacific and Caribbean, respectively) were found to be very closely related, whereas S. pulchrorum and S. corculorum from the Pacific were found to be distantly related. Analyses of 10 additional symbiotic and nonsymbiotic dinoflagellates, using partial SSU rDNA sequences to generate a tentative dinoflagellate phylogeny, indicate that members of the genus Symbiodinium cluster with most of the other (free‐living) dinoflagellates in the genus Gymnodinium. The genus Amphidinium, as represented by A. carterae and A. belauense, appear to be distantly related to the other members of the Gymnodiniaceae. This analysis, combined with morphological and biochemical data, indicates that the symbionts S. pulchrorum (from Aiptasia pulchella) and S. bermudense Trench (from Aiptasia tagetes) from the Indo‐Pacific and Caribbean, respectively, are very closely related but are not identical.

Bacterial diversity associated with the Caribbean tunicate Ecteinascidia turbinata

The Caribbean tunicate, Ecteinascidia turbinata produces the anti-cancer agent ET-743 that could well be a metabolite of an associated bacterial strain. This current study aims at the analysis of bacteria that are persistently and specifically associated with this invertebrate. Utilizing techniques such as denaturing gradient gel electrophoresis, DNA sequencing and phylogenetic analysis of bacteria from E. turbinata collected from different locations in the Caribbean Sea, we report here the identification of five possible persistently associated bacteria. Of these, only one organism, Candidatus Endoecteinascidia frumentensis, was found specifically associated to E. turbinata from the Caribbean and has also been found to be associated with E. turbinata from the Mediterranean. These experiments suggest that assessment of bacterial diversity associated with invertebrates from different geographical sites might be an effective way of identifying persistently and specifically associated bacteria.

The nucleotide sequence of the small subunit ribosomal RNA gene from Symbiodinium pilosum, a symbiotic dinoflagellate.

SummaryThe complete sequence of the small subunit ribosomal RNA (SSU rRNA) gene was determined for the symbiotic dinoflagellate Symbiodinium pilosum. This sequence was compared with sequences from two other dinoflagellates (Prorocentrum micans and Crypthecodinium cohnii), five Apicomplexa, five Ciliata, five other eukaryotes and one archaebacterium. The corresponding structurally conserved regions of the molecule were used to determine which portions of the sequences could be unambiguously aligned. Phylogenetic relationships were inferred from an analysis of distance matrices, where pair-wise distances were determined using a maximum likelihood model for transition and transversion ratios, and from maximum parsimony analysis, with bootstrap resampling. By either analytical approach, the dinoflagellates appear distantly related to prokaryotes, and are most closely related to two of the Apicomplexa, Sarcocystis muris and Theileria annulata. Among the dinoflagellates, C. cohnii was found to be more closely affiliated with the Apicomplexa than either P. micans or S. pilosum.

A semiempirical model for predicting biodegradation profiles of individual polymers in starch-poly- (?-hydroxybutyrate-co-?-hydroxyvalerate) bioplastic

Plastic prepared from formulations of cornstarch and poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV) biodegraded in tropical coastal waters. Biodegradation was monitored for 1 year. Starch - PHBV bioplastic appeared to lose weight in two overlapping phases until both biopolymers were entirely consumed. To examine the underlying degradation of starch and PHBV from biphasic weight-loss profiles, a semiempirical mathematical model was developed from which degradation profiles and lifetimes of the individual biopolymers could be predicted. The model predicted that starch and PHBV in the bioplastic had half-lives of 19 days and 158 days, respectively. Computed profiles also predicted that the starch in the composite would be completely degraded in 174 days, while residual PHBV would persist in the marine environment for 1107 days. The model further revealed that, for a 30% starch : 70% PHBV composite, PHBV degradation was delayed 46 days until more than 65% of the starch was consumed. This suggested that PHBV degradation was metabolically repressed by glucose derived from starch. Glucose repression of microbial PHBV degradation was substantiated in 91 of 100 environmental isolates. The validity of the elaborated model was proven when its revelations and predictions were later confirmed by chemical analysis of residual bioplastic samples.

Culturable and nonculturable bacterial symbionts in the toxic benthic dinoflagellate Ostreopsis lenticularis.

The toxic benthic dinoflagellate Ostreopsis lenticularis hosts a variety of symbiont bacterial flora. Laboratory cultured Ostreopsis clones require the presence of symbiotic Pseudomonas/Alteromonas bacterial strains for growth and toxicity development. Three culturable bacterial strains associated with Ostreopsis were identified as Pseudomonas/Alteromonas strain 1, Pseudomonas/Alteromonas strain 2 and Acinetobacter. Denaturing gradient gel electrophoresis (DGGE) analyses of extracted Ostreopsis associated bacterial DNAs indicated that there were three culturable and four non-culturable associated bacterial strains. The results presented here are the first report of the presence of unculturable bacterial symbionts in a toxic benthic dinoflagellate. Ostreopsis lost toxicity when exposed to elevated temperatures in the field and laboratory culture and subsequently recovered toxicity at reduced temperatures. Ostreopsis associated culturable Pseudomonas/Alteromonas bacterial strains were significantly reduced in dinoflagellate cultures exposed to elevated temperatures. The decreased toxicity of O. lenticularis exposed to elevated temperatures and their subsequent recovery of toxicity in periods of reduced thermal stress may have resulted from the effects of elevated temperature on the spectrum of culturable and unculturable bacterial species interacting with their Ostreopsis host.

Biodegradation of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by a Tropical Marine Bacterium, Pseudoalteromonas sp. NRRL B-30083

Pseudoalteromonas sp. NRRL B-30083 was isolated as the predominant PHBV-degrading organism from a tropical marine environment. In complex medium, the isolate grew well at temperatures between 23°C and 33°C, with an optimal doubling time of about 30 min. NaCl was required at concentrations between 0.2 N and 0.8 N. Optimal pH levels for growth were between pH 6.5 and pH 8.5. Liquid cultures grew modestly on PHBV as a sole carbon source under optimal conditions, although PHBV depolymerase activity was not detected.

POLYCLONAL ANTIBODIES AGAINST IRON‐SUPEROXIDE DISMUTASE FROM ESCHERICHIA COLI B CROSS‐REACT WITH SUPEROXIDE DISMUTASES FROM SYMBIODINIUM MICROADRIATICUM (DINOPHYCEAE)1

Assays for superoxide dismutases (SODs) were performed using cell‐free extracts of the symbiotic dinoflagellate Symbiodinium microadriaticum Freudenthal (emend Trench and Blank) after separation in undenatured polyacrylamide gels. Using appropriate inhibitors (KCN and H2O2) we detected the presence of Cu/Zn‐, Mn‐, and Fe‐SODs. In immunoblot assays, polyclonal antibodies against Fe‐SOD from Escherichia coli B cross‐reacted with two major polypeptides in the water‐soluble fraction and one polypeptide in the Triton X‐100‐solubilized pellet fraction. The polypeptide common to both fractions, with a relative molecular mass of 43.5 kDa, was identified as Mn‐SOD. In S. microadriaticum, FeSOD, found only in the water‐soluble fraction, appears to be monomeric, with a relative molecular mass of 49.5 kDa.

Construction of Plasmid Vectors and Transformation of the Marine Yeast Debaryomyces hansenii

Abstract We have constructed two plasmid vectors (pMR95 and pMR96) with selectable markers for the marine yeast Debaryomyces hansenii. Plasmid pMR95 contains an autonomously replicating sequence previously isolated from Debaryomyces and a hygromycin B resistance gene from the plasmid pLG90 under the control of the isocytochrome C1 promoter and terminator sequences, while pMR96 has, in addition, the Saccharomyces URA3 gene. Transformation in Debaryomyces was accomplished by electroporation. Plasmid pMR95 was capable of transforming both Saccharomyces cerevisiae and D. hansenii to hygromycin resistance at low frequencies; pMR96 transformed both yeasts at low frequencies when selected for hygromycin B resistance and at very high efficiencies when selected for uracil prototrophy. The presence of the plasmids in the transformed yeast was confirmed by polymerase chain reaction. The plasmids could be recovered back in Escherichia coli when transformed with total DNA from the yeast transformants, indicating at least a partial autonomous existence of the plasmids in the marine yeast. To our knowledge this is the first successful attempt to transform D. hansenii.

Regulated expression of green fluorescent protein in Debaryomyces hansenii

The broad range of environmental conditions under which Debaryomyces hansenii can grow, and its production of lipolytic and proteolytic enzymes, have promoted its widespread use. The present work represents a preliminary characterization of D. hansenii for heterologous expression and secretion of green fluorescent protein (GFP). Six heterologous expression vectors were used to address protein production efficiency under regulated expression conditions. Protein expression in D. hansenii seems to be similar to that in Saccharomyces cerevisiae, with transcription being controlled by almost all of the S. cerevisiae and D. hansenii inducible promoters tested, with the exception of the alcohol dehydrogenase 2 gene promoter from S. cerevisiae. Extracellular protein levels in D. hansenii were lower than in S. cerevisiae when Saccharomyces signal peptides were used.

Biodegradation of toxic chemicals in Guayanilla Bay, Puerto Rico

Studies were conducted to assess the factors that may influence the rate and extent of biodegradation of biphenyl, naphthalene, phenanthrene, pentachlorophenol (PCP) and p-nitrophenol in water samples collected from the Guayanilla Bay (18 degrees N; 67.45 degrees W), southwest of Puerto Rico. In vitro studies mediated slow degradation of biphenyl, naphthalene and phenanthrene substrates by natural microbial flora present in the Bay. Addition of KNO(3) as a source of inorganic N greatly enhanced the degradation of phenanthrene but not of naphthalene, suggesting that effects on degradation due to nutrient limitation were compound specific. The rate and extent of degradation of naphthalene and PCP were higher in water samples collected closer to the source of contamination, i.e. the petrochemical complex. The identity of a phenanthrene degrading bacterium, previously identified by conventional phenotypic method (Zaidi et al., Utilizing Natures Advanced Materials, Oxford Unviersity Press, 1999) as Alteromonas sp., was confirmed by partial DNA sequencing of the small subunit rRNA gene.