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2004 Funded Pilot Projects

Characterization of a cyanobacterial anti-algal compound

Eric Webb and Chris Reddy, Woods Hole Oceanographic Institution

Cyanobacteria are well-known, ubiquitous components of the biosphere whose most important role is in the global carbon cycle - where they remove CO₂ from the atmosphere and subsequently produce O₂. Over geologic time, this physiological activity was responsible for producing the oxygenated atmosphere that was required for human evolution and life. In addition to this important biogeochemical role, cyanobacteria have been shown to be potent producers of natural products (antibiotics, toxins, etc).

Recent work from the Webb laboratory has demonstrated that Microcoleus PCC7420, a filamentous, non-diazotrophic cyanobacterium isolated from a Woods Hole salt marsh, contains two gene families that have been implicated in natural product synthesis: non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS). Additional biochemical work has shown that this strain produces a compound(s) that inhibits the growth of other cyanobacteria. Cell-free extracts from Microcoleus PCC7420 consistently inhibited the growth of two test cyanobacterial strains.

Herein we propose to purify and structurally characterize the growth-inhibiting compound and determine the environmental cues that regulate its expression. These data and a partial genome sequence (obtained by the Sogin Laboratory) will be indispensable in identifying the genes responsible for its synthesis. Further experiments will be designed to determine if this compound is a cyanobacterial-specific inhibitory compound, a more general antimicrobial or antibiotic, or a eukaryotic and prokaryotic phytoplankton inhibitor.

Microbial natural products already serve as one of the foundations of pharmacology. Although it has been demonstrated that cyanobacteria produce natural products, due to the perceived difficulty in culturing these organisms, relatively few compounds have been characterized to date. In the future cyanobacterially-derived natural products might be used in diverse sectors of society, including medicine, agriculture, and biocontrol.

We plan to use the data from this study to obtain NSF funding to characterize the ecological significance of this compound in the salt marsh ecosystem and pursue NIH funding to characterize additional activities observed in other strains of cyanobacteria.

Cnidarian toxins against voltage-gated Ca²⁺ channels

Robert Greenberg, Marine Biological Laboratory

Cnidarians such as jellyfish and sea anemones produce venoms that are comprised of a variety of toxins. Several of these toxins have been characterized and are targeted against specific receptors and ion channels in excitable cells. For example, polypeptide sea anemone toxins have dramatic effects on voltage-gated sodium channels. They bind selectively to these channels and inhibit inactivation of the channel.

Peptide toxins from sea anemones have also been shown to selectively inhibit different types of voltage-gated potassium channels. Voltage-gated calcium (Ca²⁺) channels are critical components of excitable cells. They open in response to changes in membrane potential and regulate levels of intracellular Ca²⁺, an important second messenger. Ca²⁺ channels are targets for several agents used to treat cardiovascular diseases, and they are also being exploited as targets for pharmacological management of pain, epilepsy, and neural ischemia and stroke, as well as other conditions.

Ca²⁺ channels are also acted upon by toxins from arthropods and molluscs. Many cnidarian venoms serve to paralyze their prey, and Ca²⁺ channels associated with the neuromuscular junction may be specifically targeted by toxins found within these venoms.

This project will use heterologously expressed Ca²⁺ channels to screen cnidarian venoms for toxins that interact with these channels. Various species of cnidarians will be collected from east-coast waters, and crude venom extracts will be tested for their effects on expressed Ca²⁺ currents. If specific effects are found, the active toxin components will be isolated by standard biochemical approaches (eg, HPLC), and characterized. These experiments may provide new pharmacological tools for analysis of these channels, and may eventually lead to novel drugs for therapeutic use.

Marine phage as vectors of gene transfer between marine bacteria and bacterial pathogens

Jon King, Massachusetts Institute of Technology

Bacterial infections continue to be a major source of disease and mortality worldwide. A diverse set of genes and gene clusters necessary for bacterial pathogenesis have been carefully documented in the last decade. A subset of these virulence genes are found encoded within facultative pathogens by bacteriophages in the integrated, or lysogenic state (prophages). Conversion of avirulent bacteria to virulent forms by phage lysogeny has been demonstrated in the laboratory and inferred through genomic comparisons between closely related bacterial strains differing in pathogenicity. However, very little is known of the sources and environmental distribution of phage-encoded virulence genes or the evolutionary routes through which they have been acquired by their bacterial hosts.

Bacteriophages are extremely abundant in marine waters, with numbers approaching 107/ml. Recent genomic data suggest that phages of diverse hosts can exchange sequences within a global gene pool. This exchange is reflected in the surprising number of genetic and structural features common between phages of marine heterotrophic bacteria, phages of marine photosynthetic bacteria, and well studied phages of enteric bacteria, including the types causing human diseases. Therefore, the ocean may be an important reservoir of phageencoded virulence genes and may be an active site for phage mediated exchange of these sequences between bacterial populations.

The overall goals of this project are to investigate whether phage-borne genes or gene clusters associated with virulence effects in bacterial pathogens of humans, are also found among phages infecting marine photosynthetic bacteria.

The experiments involve:

a) Isolation of phages of marine and enteric bacteria from estuaries and coastal waters where enteric bacteria from terrestrial runoff or sewage effluents mix with coastal ocean waters carrying marine bacteria. b) Using oligonucleotide probes for known phage-borne virulence genes, assaying filtered water samples for these sequences. If detected, further investigation would determine whether they were associated with isolatable phages of either laboratory enteric bacteria and/or coastal ecotypes of the marine obligate photoautoroph, Synechococcus.

Positive results would open the possibility of identifying sources of extant as well as emerging pathogenic sequences through more systematic investigation of marine microorganisms.