Abstract:

Brominated organics comprise a largely marine group of important chemicals that mediate ecological interactions. Although they are found in terrestrial ecosystems, in plants, bacteria, fungi, lichens, insects and in some higher animals, they are most abundant and diverse in benthic marine habitats, where the precursors for their biosynthesis are readily abundant. A class of enzymes known as bromoperoxidases catalyze the biosynthesis of organobromines starting with molecular bromine, oxidized bromide, or tribromide ions as the brominating reagents. Combined with the appropriate organic, oxygen, sulfur and/or nitrogen containing compounds, they can produce bromophenols, bromopyrroles, brominated hydrocarbons, sulfides and a wide array of other organobromines. Brominated organics of marine origins appear in polychaetes, hemichordates, molluscs, crustaceans, fish and even bioaccumulate in some seabirds. Although widespread the function of this class of chemicals is poorly understood. In the pursuit of understanding the ecological relevance of these chemicals, the most heavily studied system has been that of the benthic marine worms (polychaetes & hemichordates), their predators and the microorganisms that inhabit their burrows. Early work on the organobromines secreted by these marine worms, largely bromophenols and bromopyrroles, pointed to their having both anti-microbial and anti-predation properties. This has been backed by several studies that show organobromine levels are highest in exposed portions of the worm's bodies, that sedimentary bacteria have highly developed detoxification systems for bromophenols, and that anaerobic bacterial total biomass and respiration rates are especially sensitive to bromophenols. It addition to these findings bromophenols have been shown to serve as negative recruitment cues for juvenile sedimentary dweller (polychaetes and bivalves) indicating that they may also play an important role in determining the composition of benthic assemblages. Conflicting with these early models for organobromine activity are data from several recent studies. Direct sampling of bacteria biomass (by microscopy and PLFA analysis), more data on microbial respiration rates in the presents of bromophenols and predation studies are redefining how we look at this model system. It is evident from these conflicting studies that our current understanding of brominated organics in marine ecosystems is incomplete and that interactions may be more complicated then was originally suspected.

Introduction:

Although not as well developed as the study of organochlorine compounds, organobromine compounds have become of increasing interest to science due to our increasing understanding of their abundance and biological functions. The use of organobromines as signal molecules by polychaetes, hemichordates and other marine organisms is novel in comparison to terrestrial organisms which must rely on much smaller and lighter compounds. The aquatic environment nullifies the degree of volatility needed by terrestrial signal compounds where water provides a much more forgiving solvent system then air. Not being subject to the restriction of the terrestrial/none-aquatic environment has lead to a much more diverse array of signal molecules of which the organobromines are a part (Hay, 1996). The first naturally occurring marine organobromine compound was isolated and characterized in 1909 from molluscs (Friedlander, 1909) but as recently as 1973 there were only around 60 known naturally occurring organobromine compounds (Siuda et al., 1973). That number has increased rapidly over the last 30 years and currently there are 1,690 bromine compounds that have been identified (Gribble, 2000). As with other naturally occurring organohalides, organobromines comprise a largely marine class of compounds with only a few terrestrial members. They are known from plants, fungi, lichen, algae, bacteria, insects and from some higher animals although they are most abundant in benthic and other marine habitats (Gribbles, 2000). Marine organisms such sponges, corals, sea slugs, tunicates, hemichordates, and polychaetes are abundant producers of brominated organics (Fielman et al., 1999) typified by species such as the acorn worm Ptychodera flava where the fecal secretions of 64 million worms per km² can be as high as 4 tons year^-1 km^-2 (Higa & Sakemi, 1983). These compounds have a wide impact on their ecosystem and have been found in molluscs, crustaceans, fish, and are even bioaccumulated in some seabirds (Fielman et al., 1999; Tittlemier et al, 1999). In fish, shrimp and some other commercial seafood, bromophenols are responsible for a major iodoform-like off-flavor (Whitfield et al., 1995, 1997, 1998, 1999). This has lead to considerable research by the CSIRO into the distributions of these compounds in and around Australia. Although widespread the function of this class of chemicals is poorly understood. While progress in analytical techniques has allowed us to identify large numbers of marine organobromines in the last 30 years their ecological relevance has remained somewhat unclear. This is reflection of the fact that while analysising for a particular compound can be quite straight forward, elucidating that same compound's biological activity through an appropriate bioassay can be arduous, complex and time consuming. This has been the case with benthic marine worm bromophenols and bromopyrroles. Both hemichordates and polychaetes are marine worm that live in estuaries, subtidal and intertidal areas. They modify the sediments they inhabit by their feeding, defecating, secretions and extensive burrowing. This paper will examine studies that have been done on hemichoradates and polychaetes, their predators and the microorganisms that inhabit their burrows as a model system for exploring the ecological activity of these novel organobromine compounds. The biosynthesis and molecular aspects of bromophenols will first be explored. Then a closer look will be taken at somewhat conflicting results that have arisen from the study of the marine worm system. Early work on the organobromines secreted by these marine worms, largely bromophenols and bromopyrroles, pointed to their having both anti-microbial and anti-predation properties. However recent research has put these theories into doubt.

Discussion:

An Overview of Marine Organobromine Synthesis:

Chemists in the field have termed the biosynthetic pathway for organobromines, biobromination. This process of bromination is carried out by a class of enzymes called bromoperoxidases (BPOs). BPOs have been isolated from bacteria, several marine worms and almost one hundred species of marine algae (Moore & Okuda, 1996). As their name implies BPOs use hydrogen peroxide combined with bromide and organic reactants to produce brominated organic compounds. These one step reactions have been shown to include ring and ester formation as well as simple bromination. The organic reactants run the spectrum from simple hydrocarbons to high complexity/high molecular weight compounds the products of which are shown in figure 1. Molecular bromine, oxidized bromide or tribromide ion have all been shown as suitable bromide reactants (Yang et al., 1995). The genes that code for two BPOs in Streptomyces aureofaciens have been cloned and sequenced (Pelletier et al., 1994; Pfeifer et al., 1992) giving a starting place for building an understanding of BPOs from a molecular standpoint. In addition one of the BPOs, A2, from S. aureofaciens has been characterized by X-ray crystal defraction (Hecht et al., 1994). The general topography of the enzyme was reported as was the specific atomic orientations of the area surrounding the active site. The active site itself is comprised of a catalytic triad of Ser, Asp, and His with a proposed mechanism similar to that of the model enzyme chymotrypsin. It was determined that A2 catalyzes the biobromination of substrates without assistance from cofactors such as metal ions or heme groups. In gaining a greater understanding of the molecular and chemical processes involved in biobromination we move toward a more complete understanding of organobromines role in biological systems.
 
 

Marine Worms, Their Predators and Bacteria; The case for activity:

The first work done with organobromines secreted by polychaetes and hemichordates was undertaken in the early 1960's. 2,6-dibromophenol (2,6-DBP) and 2,4,6-tribromopyrrole (2,4,6-TBP) (Fig 2.) were shown to have a deterrent effects against a wide variety of taxa. Lab tests showed fungicidal, bactericidal, ascaricidal, and molluscicidal effects (Zsolnai, 1960; Jeney & Zsolnai, 1967; Hashimoto, 1978). Following on from this work it was suggested by Higa et al. that marine worms produce these brominated compounds as defensive chemicals against microorganisms in their burrows and as deterrents to marine predators (Higa et al., 1980). This early work laid the foundation for King who in the mid 1980's published two highly recognized papers, one in Nature and one in Experientia. The first publication detailed research done with the
 
 

hemichordate Saccogloussus kowalewskii (King 1986) which is relatively common along the eastern coast of the United States. 2,4-DBP which was secreted by S. kowalewskii into its burrow walls was shown to have negative effects on microbial activity. Glucose oxidation was used as a measure of microbial activity in field tests and prepared slurries in the lab. Anaerobic microbial activity was little effected by 2,4-DBP but aerobic activity was quite sensitive. King also looked at 2,4-DBP distribution and found that it was highest in the burrow walls in a rather thin layer. S. kowalewskii uses a mucous lining to maintain the integrity of its burrow wall so it was theorized that S. kowalewskii secreted the bromophenols to prevent degradation of its burrow walls by microbial activities. These results were further supported by research, two years later, showing analytically determined concentrations of 2,4-DBP in S. kowalewskii burrow walls to be inhibitory to growth in both aerobes and anaerobes. Radiolabelled 2,4-DBP was also introduced into slurries where it was only mildly degraded by anaerobes and to a far lesser extent by aerobes (King, 1988). In a separate study anaerobic bacteria were isolated from the burrow sediments of S. kowalewskii and another hemichordate Balanoglossus aurantiacus that could preferentially remove orthobromines thereby degrading 2,4,6-TBP to 2,4-DBP and 4-bromophenol (Steward et al., 1995). NADH and NADPH added to slurries simulated increased rates of debromination suggesting a mechanism involving reduction is employed. The existence of this debromination mechanism further supported the theory that bromoorganics are toxic to microbes and that they must find detoxification methods to survive in worm burrows. Also investigated (King, 1988) were sulfate-reducing bacteria which showed little ability to degrade 2,4-DBP. 2,4-DBP decreased bacterial biomass in all tests and was considered to be toxic to the microbes (King, 1988). Sulfate reduction was further investigated along with ammonium oxidation in the presents of an expanded array of polychaete and hemichordate organobromines (4-bromophenol, 2,4-DBP, 2,6-dibromophenol (2,6-DBP), 2,4,6-tribromophenol (2,4,6-TBP), and 2,3,4-tribromopyrrole) (fig. 2) in bulk intertidal sediments and burrow wall sediments (Giray & King, 1997a). Sulfate reduction inhibition was either unaffected by <100 nmol/cm³ concentrations or did not correlate with organobromine concentrations. Ammonium oxidation was inhibited in surface sediments at concentrations as low as 1 nmol/cm³ but increased ammonium oxidation in some burrow wall sediments and decreased it in others.

When taken as a whole the studies of microbial activity in the presence of bromophenols seemed to show that they did indeed have anti-microbial activity. The one exception to this was the results from the work done on sulfate-reducing bacteria. However, this result was explained by proponents of the theory as examples of a bacterial strain gaining resistance to bromophenols.

Besides anti-microbial activity organobromines were postulated to have deterrent effects on marine predators. To this end immunofluoresence staining and immunoblotting experiments were carried out to determine the location and concentration of chloroperoxidase and its products in the polychaete Notomastus lobatus (Yoon et al., 1994). Chloroperoxidase is the primary enzyme involved in the biosynthesis of bromophenols (4-bromophenol, 2,4-DBP, and 2,4,6-TBP) in N. lobatus. Enzyme concentrations were shown to be highest in the tail, intermediate in the epidermis of the mid-body, and wholly lacking in the head region. Bromophenol concentration mirrored that of the enzyme. This result supports the postulate that N. lobatus, which feeds head down, sequesters bromophenols in its most exposed area, its tail, as a chemical deterrent against predators. A similar pattern of organobromine distribution was observed in S. kowalevskii (Fielman & Targett, 1995). Here 2,3,4-tribromopyrrole was in highest concentration (1.5% ash-free dry wt.) in the tail of the worm. Here again the marine worm is a head down feeder so the tail is the most exposed area of the body. 2,3,4-tribromopyrrole showed temporal increases which correlated with spawning suggesting increased defense at a particularly critical time where exposure to predation is greater. Using fast-atom bombardment mass spectrometry a novel sodium sulfamate salt of 2,3,4-tribromopyrrole was discovered at higher concentrations then 2,3,4-tribromopyrrole itself (11% vs. 0.6%). This sodium salt was suggested to be a precursor to 2,3,4-tribromopyrrole that is more stable and less autotoxic. Concentrations of the sodium salt were found to be highest in the hepatic region (50%) which is the presumed site for biosynthesis. Further studies to test the activity of these two compounds were suggested by the authors but these have yet to be published.

A third function has been advanced for bromophenols which is that of a negative recruitment cue. In trials where juveniles of two bivalve species and one polychaete species, Thelepus crispus, were settled in sediments contaminated with synthetic bromophenols significant deterrence was shown when compared to a control (Woodin et al., 1997). In bivalves juveniles 50% and 67% did not burrow into bromophenol treated sediment while 100% of juveniles burrowed into the control sediment. T. crispus juveniles burrowed into the treated sediments at higher rates but still showed significant differences between control and bromophenol contaminated sediments. These results suggest bromophenols play a role in determining the composition and benthic assemblages. This may be a mechanism by which juveniles can choose previously unsettled sites or undersettled sites where competition for space and resources is more limited thereby giving them a greater chance of survival.

Polychaetes, Their Predators and Bacteria; Activity questions:

Doubts about the postulated roles for bromophenols and bromopyrrole came into focus in the mid 1990's with the publication of several papers that cast suspicion on the previously proposed theories. Steward et al. published a paper in 1996 which compared the burrow linings of three marine worms, N. lobatus, B. aurantiacus, and Branchyoasychus americana, with nearby sediments from the surface and subsurface. With the doubts that had been raised about bromophenols anti-microbial properties in mind (Giray & King, 1997a) the researchers wanted to use a direct sampling assay of microbial biomass that would allow them to take multiple samples from burrows without destroying them. To do this Steward et al. employed ester-linked phospholipid fatty acid (PLFA) analysis was, which could differentiate lipid biomarker PLFA for the taxa of microbes involved. Signature PLFA blends could by used to determine differences in microbial community make-up and total biomass. Burrow wall sediments tested in this study were found to be significantly different from surface and subsurface sediments with respect to microbial biomass. However, these differences were attributed to burrow structure, the texture of burrow sediments and carbon content and not to bromophenol concentration. No differences were found by PLFA analysis in biomass between bromophenol containing burrows and those without. In addition there were no distinctive differences in PLFA signatures between burrows which would have indicated that there were separate microbial communities between burrows with and without bromophenols.

A core finding in support of bromophenols having anti-microbial properties came from the work that showed microbial respiration was inhibited by bromophenols. This finding was revisited by researchers who took core sediment samples from N. Lobatus burrow sites and from similar areas were no bromophenols were present (Lovell et al., 1999). They added the radiolabelled substrates acetate or glucose and varying concentration of 4-bromophenol to the sediment samples and incubated at in situ temperature. Baseline levels of microbial assimilation and respiration were determined for both the radiolabelled substrates and these were used as the controls for comparison with the 4-bromophenol treated sediments. No significant reductions were found in either microbial assimilation or respiration of the radiolabelled compounds even at bromophenol concentration a hundred times that of the ambient concentrations in sediments from which the samples were taken. These results are striking in their dissimilarity from those obtained by earlier researchers. They suggest that bromophenols may play little or no role in microbial control by marine worms.

These two studies were backed by others that questioned bromophenols anti-microbial activity. Sampling from N. Lobatus burrows where bromophenol concentration (4-bromophenol, 2,4-DBP, and 2,4,6-TBP) were compared with microbial biomass showed no effect in distribution or activity (Steward et al, 1992). In addition to the Steward et al. PLFA analysis two other studies were conducted (Alongi, 1985; Jensen et al., 1992) using direct microscopic counting. These studies looked at N. lobatus, S. kowalevskii and two other marine worms. They had similar findings showing no microbial reduction in burrows with significant bromophenol concentrations.

In 1997 King once again published a paper on bromophenols this time on the topic of anti-predatory effects of bromophenols (Giray & King, 1997b). In this study the predatory deterrence of 2,4-DBP was tested in lab aquaria and in the field with the carnivorous crab Pagurus longicarpus and the omnivorous polychaetes Glycera dibranchiata, Nereis virens, and Nephtys incisa. These predators are found in tidal areas where S. kowalevskii burrow and are thought to be the main predatory threats. While they were not known predators of S. kowalevskii the results of the experiment show that their selection was appropriate. P. longicarpus was observed during the course of the experiment feeding on S. kowalevskii in the field and in lab aquaria. S. kowalevskii was eaten by P. longicarpus even in preference to shrimp which are known to constitute a sizable amount of the crabs usual diet. The presumed predatory polychaetes were not observed feeding on S. kowalevskii. Observation of the polychaete's feeding behavior were difficult though as both predator and prey live within the sediment bed. However, elevated levels of 2,4-DBP in the polychaetes were associated with the disappearance of S. kowalevskii indicating that predation did indeed occur.

Conclusions:

It is evident from these conflicting studies that our current understanding of brominated organics in marine ecosystems is incomplete and that interactions may be more complicated then was originally suspected. Although conflicting, the results of the research that has been conducted on hemichordates and polychaetes and the organobromines they secrete, are not mutually exclusive. It may be that organobromines do have deterrent effects on as these some microbes and predators. However it is no longer possible, in light of the more recent research, to claim that we have an coherent theory of how this system operates. A definitive role for organobromines in the hemichordate and polychaete system remains to be shown. We have in this system an opportunity to explore the activity of novel compounds not greatly studied before. With additional information from chemical, molecular and ecological work we will be well placed to gain a greater understanding of this system.

References:

Alongi Dm Microbes, Meiofauna, And Bacterial Productivity On Tubes Constructed By The Polychaete Capitella-Capitata Marine Ecology-Progress Series 23: (2) 207-208 1985

Fielman KT, Woodin SA, Walla MD, Lincoln DE Widespread occurrence of natural halogenated organics among temperate marine infauna Marine Ecology-Progress Series 181: 1-12 1999

Fielman Kt, Targett Nm Variation Of 2,3,4-Tribromopyrrole And Its Sodium Sulfamate Salt In The Hemichordate Saccoglossus-Kowalevskii Marine Ecology-Progress Series 116: (1-3) 125-136 Jan 1995

Friedlander P Chem. Ber. 42: 765 1909

Giray C, King GM Predator deterrence and 2,4-dibromophenol conservation by the enteropneusts Saccoglossus bromophenolosus and Protoglossus graveolens Marine Ecology-Progress Series 159: 229-238 1997a

Giray C, King GM Predator deterrence and 2,4-dibromophenol conservation by the enteropneusts Saccoglossus bromophenolosus and Protoglossus graveolens Marine Ecology-Progress Series 159: 229-238 1997b

Gribble GJ The natural production of organobromine compounds Environmental Science And Pollution Research 7: (1) 37-49 2000

Hashimoto Y, Kamiya H Studies On Marine Toxins .52. Occurrence Of Saxitoxin And Related Toxins In Palauan Bivalves Toxicon 16: (3) 303-306 1978

Hay ME Marine chemical ecology: What's known and what's next? Journal Of Experimental Marine Biology And Ecology 200: (1-2) 103-134 Nov 15 1996

Hecht Hj, Sobek H, Haag T, Pfeifer O, Vanpee Kh The Metal-Ion Free Oxidoreductase From Streptomyces-Aureofaciens Has An Alpha/Beta Hydrolase Fold Nature Structural Biology 1: (8) 532-537 Aug 1994

Higa T, Sakemi Si Environmental-Studies On Natural Halogen Compounds .1. Estimation Of Biomass Of The Acorn Worm Ptychodera-Flava Eschscholtz (Hemichordata, Enteropneusta) Andexcretion Rate Of Metabolites At Kattore Bay, Kohama Island, Okinawa Journal Of Chemical Ecology 9: (4) 495-502 1983

Higa T, Fujiyama T, Scheuer Pj Halogenated Phenol And Indole Constituents Of Acorn Worms Comparative Biochemistry And Physiology B-Biochemistry & Molecular Biology 65: (3) 525-530 1980

Jeney E, Zsolnai T The antimicrobial, ascaricidal, and molluscicidal activities of salicylanidide analogs Zentraalbl. Bakteriol. 202: 547-562 1967

Jensen P, Emrich R, Weber K Brominated Metabolites And Reduced Numbers Of Meiofauna Organisms In The Burrow Wall Lining Of The Deep-Sea Enteropneust Stereobalanus-Canadensis Deep-Sea Research Part A-Oceanographic Research Papers 39: (7-8a) 1247-1253 Jul-Aug 1992

King Gm Dehalogenation In Marine-Sediments Containing Natural Sources Of Halophenols Applied And Environmental Microbiology 54: (12) 3079-3085 Dec 1988

King Gm Inhibition Of Microbial Activity In Marine-Sediments By A Bromophenol From A Hemichordate Nature 323: (6085) 257-259 Sep 18 1986

Lovell CR, Steward CC, Phillips T Activity of marine sediment bacterial communities exposed to 4-bromophenol, a polychaete secondary metabolite Marine Ecology-Progress Series 179: 241-246 1999

Moore CA, Okuda RK Bromoperoxidase activity in 94 species of marine algae Journal Of Natural Toxins 5: (3) 295-305 OCT 1996

Pelletier I, Pfeifer O, Altenbuchner J, Vanpee Kh Cloning Of A 2nd Nonheme Bromoperoxidase Gene From Streptomyces-Aureofaciens Atcc-10762 - Sequence-Analysis, Expression In Streptomyces-Lividans And Enzyme-Purification Microbiology-Uk 140: 509-516, Part 3 Mar 1994

Pfeifer O, Pelletier I, Altenbuchner J, Vanpee Kh Molecular-Cloning And Sequencing Of A Nonheme Bromoperoxidase Gene From Streptomyces-Aureofaciens Atcc-10762 Journal Of General Microbiology 138: 1123-1131, Part 6 Jun 1992

Siuda Jf, Debernar.Jf Naturally Occurring Halogenated Organic Compounds Lloydia-The Journal Of Natural Products 36: (2) 107-143 1973

Steward CC, Lovell CR Respiration and assimilation of 4-bromophenol by estuarine sediment bacteria Microbial Ecology 33: (3) 198-205 May-Jun 1997

Steward CC, Nold SC, Ringelberg DB, White DC, Lovell CR Microbial biomass and community structures in the burrows of bromophenol producing and non-producing marine worms and surrounding sediments Marine Ecology-Progress Series 133: (1-3) 149-165 MAR 1996

Steward Cc, Dixon Tc, Chen Yp, Lovell Cr Enrichment And Isolation Of A Reductively Debrominating Bacterium From The Burrow Of A Bromometabolite-Producing Marine Hemichordate Canadian Journal Of Microbiology 41: (7) 637-642 Jul 1995

Steward Cc, Pinckney J, Piceno Y, Lovell Cr Bacterial Numbers And Activity, Microalgal Biomass And Productivity, And Meiofaunal Distribution In Sediments Naturally Contaminated With Biogenic Bromophenols Marine Ecology-Progress Series 90: (1) 61-71 Dec 1992

Tittlemier SA, Simon M, Jarman WM, Elliott JE, Norstrom RJ Identification of a novel C10H6N2Br4Cl2 heterocyclic compound in seabird eggs. A bioaccumulating marine natural product? Environmental Science & Technology 33: (1) 26-33 JAN 1 1999

Whitfield FB, Drew M, Helidoniotis F, Svoronos D Distribution of bromophenols in species of marine polychaetes and bryozoans from eastern Australia and the role of such animals in the flavor of edible ocean fish and prawns (shrimp) Journal Of Agricultural And Food Chemistry 47: (11) 4756-4762 NOV 1999

Whitfield FB, Helidoniotis F, Shaw KJ, Svoronos D Distribution of bromophenols in species of ocean fish from eastern Journal Of Agricultural And Food Chemistry 46: (9) 3750-3757 SEP 1998

Whitfield FB, Helidoniotis F, Shaw KJ, Svoronos D Distribution of bromophenols in Australian wild-harvested and cultivated prawns (shrimp) Journal Of Agricultural And Food Chemistry 45: (11) 4398-4405 NOV 1997

Whitfield Fb, Helidoniotis F, Svoronos D, Shaw Kj, Ford Gl The Source Of Bromophenols In Some Species Of Australian Ocean Fish Water Science And Technology 31: (11) 113-120 1995

Woodin SA, Lindsay SM, Lincoln DE Biogenic bromophenols as negative recruitment cues Marine Ecology-Progress Series 157: 303-306 1997

Yang Zp, Shelton Kd, Howard Jc, Woods Ae Mechanism Of The Chloroperoxidase-Catalyzed Bromination Of Tyrosine Comparative Biochemistry And Physiology B-Biochemistry & Molecular Biology 111: (3) 417-426 Jul 1995

Yoon Ks, Chen Yp, Lovell Cr, Lincoln De, Knapp Lw, Woodin Sa Localization Of The Chloroperoxidase Of The Capitellid Polychaete Notomastus-Lobatus Biological Bulletin 187: (2) 215-222 Oct 1994

Zsolnai T Versuche zua entdeckung neuer fungistatika-I Phenol-derivate Biochem. Pharmacol. 5:1-19 1960