Molecular Level – To identify the cellular “receptor(s)” that ZA binds and/or interferes the binding with the originally intended “signal(s)”,

Cellular Level – To examine the effects of ZA on cell metabolism and other more specific receptor-mediated metabolic events through functional genomics and proteomics approaches,

Biofilm Level – To investigate the effects of ZA on both biological development and morphological properties of biofilms:

      Biological Development – To investigate the changes in metabolism of cells in biofilm, with ZA added at different stages of biofilm development,

      Morphological Properties – To examine biofilms for the distribution and shape of the cells and the structure variations.

Zosteric acid (ZA), a novel natural antifoulant, deters attachment of many microorganisms, algae, barnacles and tubeworms studied at sub-lethal doses.  Our earlier studies (supported by Ohio Sea Grant) further suggested it functions via interaction with cells.  However, nothing is known about the nature and mechanisms of the interactions between ZA and organisms, or how the interactions affect biofouling and biofilm development.  Gaining a better understanding on these issues is the goal of this study, and the goal is to be achieved by three levels: molecular, cellular, and biofilm, of study.  On the molecular level, the cellular “receptor(s)” that ZA binds and/or interferes the binding with the originally intended “signal(s)” will be identifiied.  On the cellular level, the effects of ZA on cell metabolism, including growth, synthesis of extracellular proteins and polysaccharides, and other more specific receptor-mediated metabolic will be examined.  On the biofilm level, the effects of ZA on both biological development and morphological properties of biofilms will be evaluated.   

Biofouling and biofilm formation are ubiquitous in nature and have significant environmental and industrial impacts.  For maritime industries, biofouling increases fuel consumption and maintenance costs of marine vessels, and leads to surface corrosion and structural damage for aquatic infrastructures.  Traditionally, heavy metal based anti-fouling paints were used to kill off the unwanted fouling organisms.  Environmental concerns on the release of these metals have resulted in their ban.  Organic biocides have been sough as alternatives; however, most of these biocides are also toxic and not just to the target organisms.  Another direction is to use foul-release coatings, but regular cleanings are required to remove soft-foulers that settle during the early stages of fouling.  Once hard foulers are formed on such coatings, their removal is difficult or impossible without damaging the coatings.  The use of non-toxic/less toxic marine product antifoulants has recently been suggested as a promising approach, especially when incorporated into foul-release coatings to discourage soft-foulers.  For these antifoulants to be utilized to their full potential, their mechanistic action against fouling organisms needs to be well understood.  However, such mechanism studies have rarely been reported, especially from the combined perspectives of biological, physiological, morphological, and physical.

The antifoulant to be used is zosteric acid, which was originally isolated and structure-identified from the extract of eelgrass Zostera marina.  We, however, have acquired the ability to synthesize and purify it to > 96%. Synthetic ZA will be used throughout this study for better quality consistency.  A Gram-negative bacterium, Pseudomonas aeruginosa PAO1, commonly found in water, soil and fouling communities and a model organism used in numerous studies on biofilm development will be utilized.  P. aeruginosa is also one of the few microorganisms with a completely sequenced genome and readily available tools (e.g. microarrays, genome/proteome databases) for analyzing metabolic networks. These tools will greatly facilitate the proposed identification of the receptor(s) bound or interfered by ZA, and elucidation of the potential receptor-mediated metabolic events.

The specific tasks to be carried out in this multileveled study are described briefly below:

Task 1 (Molecular Level) Identify cellular “receptor(s)” bound or interfered by ZA: Earlier studies indicated that free floating ZA molecules deter bacterial attachment.  The most possible mechanism for such behavior is the binding of ZA with certain receptor(s) on the bacterial surface.  To identify the putative receptor(s), P. aeruginosa will be treated with ZA at sub-lethal concentrations, and total protein lysates from treated cells will be fractionated to identify ZA bound complexes. Component of the ZA bound complexes will be further analyzed (through mass spectrometry and comparison to existing proteome databases) to identify putative receptors.

Task 2 (Cellular Level) Locate potential metabolic events affected by binding of ZA with the receptor(s): cDNA micro arrays will be used on zosteric acid treated and untreated control bacterial cells to identify differentially expressed gene networks (especially genes coding for metabolic proteins). PCR based and Western blot analysis based methods will be used to confirm the regulation of expression of the identified differentially expressed gene products

Task 3 (Cellular Level) Examine effects of ZA on cell metabolism in aqueous medium: Parameters to be investigated may include cell growth, synthesis of extracellular proteins and polysaccharides, and other more specific metabolic events triggered by the identified receptor(s) as noted in task 2.  The study will be conducted in batch culture using the common glucose-based medium added with various pertinent concentrations of ZA.  Periodic samples will be taken and analyzed to determine the profiles of cell growth and synthesis of extracellular proteins and polysaccharides. The degradation of ZA (due to variation in fluorescence intensities) by the biological system will also be followed.

The morphological structure of the biofilm on the glass slides will be imaged via the optical microscopy.  The structure variations of the biofilms, as a function of time after adding ZA to the aqueous solution, will be monitored.  The shape and distribution of P. aeruginosa (stained) in the biofilm will be examined.  Greater details of the biofilm structure will be achieved using an environmental scanning electron microscope.  Physical parameters, such as surface coverage, density, thickness, and porosity of the biofilm will also be determined.

Biofouling and biofilm formation are ubiquitous in nature and have significant environmental and industrial impacts.  For maritime industries, biofouling increases fuel consumption and maintenance costs of marine vessels [1, 2].  The associated fuel penalties cost US Navy around $100 million annually [3].  The estimated cost for removing organisms from ship-hulls, platforms and pipelines is in billions of dollars per year [1].  Biofouling causes surface corrosion and structural damage for aquatic infrastructures (e.g. oil platforms, water transportation and storage equipment, power plants), which leads to health and safety concerns.  Reducing biofouling would also eradicate the potential transfer of marine community(s) from one location to another, thereby maintaining a natural ecosystem.

Traditional heavy metal based anti-fouling paints, containing tin, copper, zinc, cadmium, chromium, released toxic materials that prompted environmental concerns, and their usage has been limited in many countries, there is an urgent need for alternative antifouling coatings.  The use of non-toxic or less toxic marine products as antifoulants represents a promising new approach. For these marine natural antifoulants to be utilized to their full potential, their mechanistic action against fouling organisms needs to be well understood.  This study is intended to understand antifouling mechanisms of a marine natural product, zosteric acid (ZA).  The fundamental knowledge on how ZA interacts with cells will markedly improve our ability in synthesizing other antifoulants and/or antibiotics with ZA-derived compounds to combat biofouling.