Perry F. Churchill

SUCCESSIONAL CHANGES IN BACTERIAL ASSEMBLAGE STRUCTURE DURING EPILITHIC BIOFILM DEVELOPMENT

Although bacteria are often treated as one entity in ecological studies, bacterial assemblages are composed of individual species populations. Bacterial assemblages can have their own richness and structure, analogous to communities of plants and animals, although few studies have attempted to describe spatial or temporal patterns in their structure. In this study, we examined successional changes in the structure of bacterial assemblages using denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction amplified 16S rDNA fragments. Bacterial biofilm assemblages developing on glass slides in a mesocosm and a small lake showed an initial increase in richness over the first week, followed by a slight decrease and a subsequent increase after two to three months. Functional changes in the bacterial community were examined using most probable number estimates and revealed decreases in the abundance of glucose- and cellulose-degraders during biofilm development, whereas benzoate-degraders became more abundant in the lake biofilms. The banding patterns observed on DGGE gels were used to derive rank-abundance profiles for each stage of biofilm development. These profiles resembled those observed for communities of macroorganisms and could usually be described by geometric series models. These models suggested greater equitability in bacterial community structure as the biofilms developed. A comparison of two successional series of biofilms separated by 30 d revealed that neither successional stage nor time of sampling was the major factor influencing bacterial assemblage structure. Our results allowed us to suggest a general model for the development of bacterial biofilm assemblages that emphasizes the interaction of species and resource diversity. This model suggests that, at least in biofilms, bacterial assemblages may not be structured by the resource competition or niche-driven patterns typical of communities of macroorganisms.

Ultrastructural and molecular analyses of Rhizophydiales (Chytridiomycota) isolates from North America and Argentina

The Rhizophydiales is the most recently circumscribed order in the Chytridiomycota. Past studies focused on soil chytrids from North America and Australia to determine the range of diversity within this clade of chytrids and established three families (Rhizophydiaceae, Terramycetaceae, and Kappamycetaceae) in the new order. Although Rhizophydiales contains seemingly simple chytrids morphologically, analyses of ribosomal gene sequences and zoospore characters have demonstrated unexpected genetic and ultrastructural diversity, highlighting the need for broader habitat and geographic sampling to reveal the actual diversity within this new order. To enlarge our sampling, in this study we investigated 38 newly cultured chytrids collected from aquatic habitats in Argentina, a territory under-explored for chytrid diversity. From analyses of thallus morphology, zoospore ultrastructure, and 28S and ITS1-5.8S-ITS2 ribosomal gene sequences, we expand the concept of Rhizophydiales, describing seven new families (Alphamycetaceae, Angulomycetaceae, Aquamycetaceae, Globomycetaceae, Gorgonomycetaceae, Pateramycetaceae, and Protrudomycetaceae) and eight new genera (Alphamyces, Angulomyces, Aquamyces, Globomyces, Urceomyces, Gorgonomyces, Pateramyces, and Protrudomyces). Results of this study underscore that even more extensive sampling from varied habitats and geographies is needed to adequately ascertain the diversity of chytrids in the Rhizophydiales.

Surfactant-enhanced bioremediation

This study was undertaken to examine the effect of three structurally related, non-ionic surfactants, Triton X-45, Triton X-100 and Triton X-165, as well as the oleophilic fertilizer, Inipol EAP 22, on the rate of biodegradation of phenanthrene by pure bacterial cultures. Each surfactant dramatically increased the apparent aqueous solubility of phenanthrene. Model studies were conducted to investigate the ability of these surfactants to enhance the rate of transport and uptake of polycyclic aromatic hydrocarbons into bacterial cells, and to assess the impact that increasing the aqueous solubility of hydrocarbons has on their rate of biodegradation. Our results indicate that increasing the apparent aqueous solubility of hydrocarbons can lead to enhanced biodegradation rates by two Pseudomonas saccharophila strains. However, our experiments also suggest that some surfactants can inhibit aromatic hydrocarbon biodegradation by certain bacteria. Our data also support the hypothesis that surface-active components present in the oleophilic fertilizer formulation, Inipol EAP 22, may have significantly contributed to the positive results reported in tests of remedial agent impact on bioremediation, which was used as a supplemental clean-up technology on Exxon Valdez crude oil-contaminated Alaskan beaches.

Rhizophlyctidales—a new order in Chytridiomycota

Rhizophlyctis rosea (Chytridiomycota) is an apparently ubiquitous, soil-inhabiting, cellulose-degrading chytrid that is the type for Rhizophlyctis. Previous studies have revealed multiple zoospore subtypes among morphologically indistinguishable isolates in the R. rosea complex sensu Barr. In this study we analysed zoospore ultrastructure and combined nu-rRNA gene sequences (partial LSU and complete ITS1-5.8S-ITS2) of 49 isolates from globally distributed soil samples. Based on molecular monophyly and zoospore ultrastructure, this group of Rhizophlyctis rosea-like isolates is designated as a new order, the Rhizophlyctidales. Within the Rhizophlyctidales are four new families (Rhizophlyctidaceae, Sonoraphlyctidaceae, Arizonaphlyctidaceae, and Borealophlyctidaceae) and three new genera (Sonoraphlyctis, Arizonaphlyctis, and Borealophlyctis).

The early-onset torsion dystonia-associated protein, torsinA, displays molecular chaperone activity in vitro

TorsinA is a member of the AAA+ ATPase family of proteins and, notably, is the only known ATPase localized to the ER lumen. It has been suggested to act as a molecular chaperone, while a mutant form associated with early-onset torsion dystonia, a dominantly inherited movement disorder, appears to result in a net loss of function in vivo. Thus far, no studies have examined the chaperone activity of torsinA in vitro. Here we expressed and purified both wild-type (WT) and mutant torsinA fusion proteins in bacteria and examined their ability to function as molecular chaperones by monitoring suppression of luciferase and citrate synthase (CS) aggregation. We also assessed their ability to hold proteins in an intermediate state for refolding. As measured by light scattering and SDS-PAGE, both WT and mutant torsinA effectively, and similarly, suppressed protein aggregation compared to controls. This function was not further enhanced by the presence of ATP. Further, we found that while neither form of torsinA could protect CS from heat-induced inactivation, they were both able to reactivate luciferase when ATP and rabbit reticulocyte lysate were added. This suggests that torsinA holds luciferase in an intermediate state, which can then be refolded in the presence of other chaperones. These data provide conclusive evidence that torsinA acts as a molecular chaperone in vitro and suggests that early-onset torsion dystonia is likely not a consequence of a loss in torsinA chaperone activity but might be an outcome of insufficient torsinA localization at the ER to manage protein folding or trafficking.

Molecular phylogenetic and zoospore ultrastructural analyses of Chytridium olla establish the limits of a monophyletic Chytridiales

Chytridium olla A. Braun, the first described chytrid and an obligate algal parasite, is the type for the genus and thus the foundation of family Chytridiaceae, order Chytridiales, class Chytridiomycetes and phylum Chytridiomycota. Chytridium olla was isolated in coculture with its host, Oedogonium capilliforme. DNA was extracted from the coculture, and 18S, 28S and ITS1-5.8S-ITS2 rDNA were amplified with universal fungal primers. Free swimming zoospores and zoospores in mature sporangia were examined with electron microscopy. Molecular analyses placed C. olla in a clade in Chytridiales with isolates of Chytridium lagenaria and Phlyctochytrium planicorne. Ultrastructural analysis revealed C. olla to have a Group II-type zoospore, previously described for Chytridium lagenaria and Phlyctochytrium planicorne. On the basis of zoospore ultrastructure, family Chytridiaceae is emended to include the type of Chytridium and other species with a Group II-type zoospore, and the new family Chytriomycetaceae is delineated to include members of Chytridiales with a Group I-type zoospore.

A molecular phylogenetic evaluation of the Spizellomycetales

Order Spizellomycetales was delineated based on a unique suite of zoospore ultrastructural characters and currently includes five genera and 14 validly published species, all of which have a propensity for soil habitats. We generated DNA sequences from small (SSU), large (LSU) and 5.8S ribosomal subunit genes to assess the monophyly of all genera and species in this order. The 53 cultures analyzed included isolates on which all described species were based, plus other spizellomycetalean cultures. Phylogenetic placement of these chytrids was explored with maximum parsimony and maximum likelihood analyses, both of which yielded comparable topologies. Kochiomyces, Powellomyces and Triparticalcar were monophyletic, while Gaertneriomyces and Spizellomyces were polyphyletic. Isolates, distinct from described species, clustered among each of the five genera, indicating that species diversity in genera is greater than currently recognized. One isolate formed a clade that included no described species, representing a new genus. Zoospore ultrastructural features and architecture seem to be good indicators of phylogenetic relationships, but finer scrutiny of characters such as kinetosome-associated structures (KAS) is needed to understand more clearly the diversity within this order as it is revised.

Succinyl-CoA : 3-ketoacid coenzyme A transferase (SCOT) : Development of an antibody to human SCOT and diagnostic use in hereditary SCOT deficiency

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) is a key enzyme for ketone body utilization. Hereditary SCOT deficiency in humans (McKusick catalogue number 245050) is characterized by intermittent ketoacidotic attacks and permanent hyperketonemia. Since previously-available antibody to rat SCOT did not crossreact with human SCOT, we developed an antibody against recombinant human SCOT expressed in a bacterial system. The recombinant SCOT was insoluble except under denaturing conditions. Antibody raised to this polypeptide recognized denatured SCOT and proved useful for immunoblot analysis. On immunoblots, SCOT was easily detectable in control fibroblasts and lymphocytes but was detected neither in fibroblast extracts from four SCOT-deficient patients, nor in lymphocytes from two SCOT-deficient patients. These data indicate that immunoblot analysis is useful for diagnosis of SCOT deficiency in combination with enzyme assay.

The microtubule-associated protein, NUD-1, exhibits chaperone activity in vitro

Regulation of cell division requires the concerted function of proteins and protein complexes that properly mediate cytoskeletal dynamics. NudC is an evolutionarily conserved protein of undetermined function that associates with microtubules and interacts with several key regulators of mitosis, such as polo-kinase 1 (Plk1) and dynein. NudC is essential for proper mitotic progression, and homologs have been identified in species ranging from fungi to humans. In this paper, we report the characterization of the Caenorhabditis elegans NudC homolog, NUD-1, as a protein exhibiting molecular chaperone activity. All NudC/NUD-1 proteins share a conserved p23/HSP20 domain predicted by three-dimensional modeling [Garcia-Ranea, Mirey, Camonis, Valencia, FEBS Lett 529(2–3):162–167, 2002]. We demonstrate that nematode NUD-1 is able to prevent the aggregation of two substrate proteins, citrate synthase (CS) and luciferase, at stoichiometric concentrations. Further, NUD-1 also protects the native state of CS from thermal inactivation by significantly reducing the inactivation rate of this enzyme. To further determine if NUD-1/substrate complexes were productive or simply “dead-end” unfolding intermediates, a luciferase refolding assay was utilized. Following thermal denaturation, rabbit reticulocyte lysate and ATP were added and luciferase activity measured. In the presence of NUD-1, nearly all of the luciferase activity was regained, indicating that unfolded intermediates complexed with NUD-1 could be refolded. These studies represent the first functional evidence for a member of this mitotically essential protein family as having chaperone activity and facilitates elucidation of the role such proteins play in chaperone complexes utilized in cell division. C. elegans NUD-1 is a member of an evolutionary conserved protein family of unknown function involved in the regulation of cytoskeletal dynamics. NUD-1 and its mammalian homolog, NudC, function with the dynein motor complex to ensure proper cell division, and knockdown or overexpression of these proteins leads to disruption of mitosis. In this paper, we show that NUD-1 possesses ATP-independent chaperone activity comparable to that of small heat shock proteins and cochaperones and that changes in phosphorylation state functionally alter chaperone activity in a phosphomimetic NUD-1 mutant.

Inhibition of mitochondrial respiration by cationic rhodamines as a possible teratogenicity mechanism

Exposure of mice to cationic rhodamines, Rh 123 and Rh 6G, has been found to be associated with developmental toxicity, while neutral rhodamines (e.g., Rh B) had no such effect. When mouse embryos from dams given ip injections of Rh 123, Rh 6G (15 mg/kg), or Rh B (30 mg/kg) on gestation Day (GD) 10 were examined, Rh 123, Rh 6G were present in embryonic tissue in fluorescent bodies within the average dimensions of mitochondria. Rh B was evenly distributed in the cytoplasm. With in vitro exposure of isolated mitochondria to rhodamines on GD 12, 3-4 times more Rh 123 was associated with mitochondria under energized conditions than under nonenergized conditions; the amount of Rh 6G associated with mitochondria was much less under either condition. Treatment of pregnant mice (ip) with Rh 123 (15 mg/kg/day) or Rh 6G (0.5 mg/kg/day) on GD 7-10 resulted in inhibition of state 3 respiration of embryonic mitochondria isolated on GD 12. When isolated embryonic mitochondria were exposed to the cationic rhodamines, inhibition of state 3 respiration was dose dependent. With 5 micrograms of Rh 123/mg mitochondrial protein, state 3 respiration decreased by 31%, while Rh 6G (1 microgram/mg) decreased state 3 respiration by 27%. In vivo exposure of maternal liver mitochondria to cationic rhodamines did not result in inhibition of respiration 2 days later, whereas in vitro results were similar to those for embryonic mitochondria. In vivo or in vitro exposure to Rh B had no effects on mitochondrial respiration. These results indicate that interference with embryonic energy metabolism is a possible mechanism by which cationic rhodamines exert adverse effects on embryogenesis.