Secondary Metabolites From Marine Bacteria

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STRATEGIES FOR THE DISCOVERY OF SECONDARY METABOLITES FROM MARINE

Annu. Rev. Microbiol. 1994.48:559-584. Downloaded from www.annualreviews.org by University of New South Wales on 03/07/11. For personal use only.
BACTERIA: Ecological Perspectiyes

Paul R. Jensen and William Fenical
Scripps Institution of Oceanography, La Jolla, California 92093-0236
KEY WORDS: bacterial ecology, biological activity, marine natural products chemistry
CONTENTS
INTRODUCTION
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560
ECOLOGICAL CONSIDERATIONS ...... .... . .. . . ... . . .... . .. . . . .. . . .. . . . . Prokaryotic Diversity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Distribution o Bacteria in the Sea .. ... . . . ......... . ... . . . . .. . . .. . .. . . f The Associations o Bacteria with Plants and Invertebrates . . . . . .. . . . . . . . . . .. . . f The Role o Symbiotic Bacteria ill f Effects o Marille Metabolites UpOIl the Distributioll o Bacteria . . .. . .. .... . .. . . f f The Adaptations o Bacteria to Diverse Maril f l Bacterial Culturability. . . . . . ..... . .. . .. . . . .. . . ... . . . . .. . . .. . . .. . . .. . .. . . The Distinctioll Betweell Marine alld Terrestrial Bacteria . ......... .... ..... . . BIOLOGICAL ACTIVITIES AND NOVEL ORGANIC SUBSTANCES............ CONCLUDING REMARKS. . . . . . . . .. . ..... . . .. . . . .. . . . .... . . . . . . . . . . . . .. . .
563
561
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566 567 569 570 571 572 574 576
ABSTRACf
Marine microorganisms have become an important point of study in the search for novel microbial products. Today, both academic and industrial interest in marine microorganisms is on the rise, in part because of the growing number of unique, biologically active secondary metabolites reported from marine bacteria. OUf ability to assess the biosynthetic potential of marine bacteria, however, is inevitably coupled to our basic understanding of their biology. This review summarizes the chemical discoveries and biological activities
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reported from marine bacteria and focuses upon several microbiological topics as they relate to natural product discovery, including the distributions, diver sity, and culturability of marine bacteria, as well as the role of symbiotic bacteria in the production of substances previously ascribed to other sources.
INTRODUCTION
Historically, the study of natural products has focused upon the elaborate biosynthetic pathways of terrestrial plants and microorganisms. In the late
1960s, however, the search for novel metabolites took a new direction as the
realm of exploration expanded to include plants and animals in the sea. This avenue of research was initiated primarily by academicians and facilitated by the development of scuba, which provided an effective means to collect shal low-water marine organisms. Today, as the result of extensive research, the study of marine natural products is recognized as both an integral component of natural products chemistry and a significant resource for drug discovery. In addition, the successes of marine natural products chemistry have helped nurture the growing discipline of marine chemical ecology, an area of research that has contributed significantly to our understanding of the ecological roles of marine secondary metabolites (see 142). The discovery that marine organisms represent a rich source of previously undescribed secondary metabolites is not entirely surprising considering that many algal and invertebrate phyla reside exclusively in the sea. In hindsight, this discovery seems only logical given that many soft-bodied marine organ isms, lacking obvious structural defenses, produce toxic secondary metabolites as a means of chemical defense. Although our understanding of chemical defense and other ecological roles of marine secondary metabolites has just begun, chemical studies of marine organisms have been extensive, yielding thousands of unique metabolites featuring new structural classes, functional groups, and a relatively high incidence of halogenation. Descriptions of the chemical discoveries in this field can be found in periodically updated reviews from
1977 to date (36, and previous reviews cited therein).
As the chemical prolificacy of marine organisms became apparent, so did their industrial potential, and marine metabolites are now being screened extensively for pharmacological activity. The results of these efforts have led to several significant biomedical discoveries. These include the clinical evalu ation of at least two marine natural products as anticancer agents-bryostatin United States, and didemnin B (150), which recently completed phase
I (143,167), which is currently in clinical trials in both Great Britain and the I clinical trials in the United States (see 77). For descriptions of the biological activities
of various marine plant and invertebrate taxa (151), as well as detailed reviews
SECONDARY METABOLITES FROM MARINE B ACTERIA
561
of the biomedical potential and pharmacological properties of marine metabo lites, a number of reviews are available
(3, 38).
Continuing efforts to obtain chemically unique marine organisms have led to the use of new and innovative methods of sample collection. These efforts include travel to far reaches of the globe to collect previously unstudied species, the use of deep-diving submersibles to access habitats that lie beyond the depths of conventional scuba, and the collection of organisms that were previously overlooked owing to difficulties in obtaining adequate biomass for chemical evaluation. These efforts have accompanied a growing interest in the study of marine bacteria, a logical and potentially productive avenue of re search given the historical significance of secondary metabolites from terres trial bacteria and the renewable nature of culturable resources. During the past decade, studies of secondary metabolites from marine bac teria have begun in laboratories throughout the world. In the most simplified sense, these studies use standard dilution and plating techniques to isolate bacteria from marine samples. Isolated bacteria are cultured in shake-flasks, on agar plates, or in fermentors, and the organic components of these cultures are then extracted, usually with organic solvents. The extracts are tested for biological activities, e.g. standard antibiotic disc diffusion assays, and analyzed for the presence of unusual chemical constituents. Organic materials of interest are purified and their structures analyzed, a complex process that generally requires sophisticated NMR methodologies. Because of growing interest in the study of secondary metabolites from marine bacteria, we would like to address in this review several topics in microbial ecology that are relevant to natural product discovery. As these topics are broad in scope and fundamental to marine microbiology, our goal is to focus upon the aspects that pertain to natural product discovery. Rather than discuss in detail chemical discoveries from marine bacteria, we refer the reader to recent reviews oriented toward this objective
(5, 39, 40, 87, 131) and offer
a summary of the biological activities and novel secondary metabolites re ported to date from marine bacteria. All of the following discussions of marine bacteria exclude cyanobacteria (unless otherwisc indicated), which are none theless an important resource for unique secondary metabolites
(36, and pre
vious reviews cited therein). In addition, one should note that novel secondary metabolites have been reported from other taxonomic groups of marine mi croorganisms, including fungi and microalgae; however, these groups are not discussed here.
ECOLOGICAL CONSIDERATIONS
By extending the search for novel microbial metabolites to the sea, a funda mental question must be addressed, i.e. do marine bacteria produce previously
562 Table 1
JENSEN & FENICAL
Novel organic molecules reported from marine bacteria
Producing strain Actinomycete
Source
Biomedical activity
References
Chaina purpurogena Streptomyces tenjimariensis Streptomyces griseus Streptomyces sioyaensis

Maduromycete
Sediment Sediment Sediment Sediment Sediment Sediment Sediment Sediment Gorgonian soft coral Jellyfish Sponge
Antibiotic, anticancer Antibiotic Antibiotic Anticancer Antibiotic Antibiotic Antibiotic No activity reported Anticancer, antibiotic
86, 136 67, 132
120, 135, 157

1 68, 169 141 140 139 138 173 1 79 76
Streptomyces sp.
Actinomycete Actinomycete Actinomycete
Streptomyces sp. Streptomyces sp.

Nonactinomycete
Antibiotic, antiinflammatory
Antibiotic
Pseudomonas bromoutilis (Alteromonas sp.) Chromobacterium (Altermonas) sp.

Pseudomonad
Seagrass Seawater Tide pool seawater Salt marsh sediment Not reported Seawater Deep-sea sediment Sponge Deep-sea sediment Fish Seawater Sponge
Antibiotic Antibiotic Antibiotic
18. 107
2
198 49 65 121, 122 58 159 85, 170 79 59 166 1 23
Beneckea gazogenes (Vibrio) Altermonas ruba

Unidentified Unidentified gram-positive species
No activity reported Bronchodilator

No activity reported Anticancer, antiviral Anticancer Anticancer No activity reported Enzyme inhibition
Alteromonas sp. Alteromonas haloplanktis Vibrio anguillarum Alteromonas sp. Alteromonas sp. Pseudomonas sp. Pseudomonas flourescens
Unidentified gram-positive species
Antimicrobial
Anticancer
Ascidian
Deep-sea sediment Hydrothermal Deep-sea sediment Seawater
Antimicrobial Anticancer
Antifungel Anticancer No activity reported
124
26 149, 1 52 178 149
Thermococcus Bacillus Alteromonas luteoviolacea
undescribed s econdary metabolites, and if so, are environmental factors spe cific to the ocean required for the production of these substances? This question is reminiscent of one fundamental to the development of marine microbiology as a scientific discipline: that is, do marine bacteria represent unique species or merely terrestrial strains adapted to the conditions of the sea? In response to the latter question, the existence of specific marine bacteria was documented nearly 50 y ears ago (see 108). The ability of marine bacteria to produce previously undescribed secondary metabolites has also been ade quately established (Table 1). Concerning the conditions necessary for the production of secondary metabolites by marine bacteria,the isolation of a novel metabolite does not in itself preclude the biosynthesis of this same substance
SECONDARY METABOLITES FROM MARINE BACTERIA
563
by a nonmarine strain. However, if this substance is produced only when conditions specific to the marine environment are provided, e.g. seawater, increased hydrostatic pressure, or a marine nutrient, the likelihood of this substance being produced as a nonmarine fermentation product is low. More over, if this substance possesses features rarely observed with metabolites from terrestrial bacteria, such as the presence of halogen atoms, the argument for its unique marine nature is further strengthened. From a natural products perspective, marine bacteria remain a relatively unexplored resource for novel secondary metabolites. Further investment in this area, from both an academic and industrial perspective, will ultimately determine the utility of this resource. To maximize the effectiveness of future research, a better understanding of marine bacterial ecology is essential. This understanding can be obtained through continued studies in, for example, the diversity, distribution, and culturability of marine bacteria, and the adaptations of bacteria for survival in both normal and extreme marine environments.
Prokaryotic Diversity
Bacterial taxonomic and metabolic diversity are fundamentally interrelated and therefore important to natural product discovery. In marine systems, how ever, we are only beginning to understand the true extent of prokaryotic diversity. This fact is adequately demonstrated by the discoveries of abundant prochlorophytes
(22), cyanobacteria (189), and most recently, archaea (28, 42)
inhabiting the world's open oceans. These discoveries, in part accounted for by the application of techniques in molecular biology to the study of microbial ecology, confirm previous beliefs that bacterial diversity in marine systems is far greater than can be accounted for by the culture-dependent techniques typically used to describe bacterial communities. As studies of prokaryotic diversity continue, the knowledge gained can be used to assess the degree to which diverse members of the marine prokaryotic community can be obtained in culture and included in the search for novel microbial products. The observation that taxonomically diverse prokaryotes have not been given adequate consideration in chemical studies of marine bacteria is supported by the relatively few genera from which novel molecules have been reported (Table
1). Are these the only taxa that produce unique metabolites, or are they
simply the only taxa studied to date? Most research in this field seems to have concentrated upon marine-derived actinomycetes and gram-negative, het erotrophic bacteria capable of growth on nutrient-rich media. This approach is logical as the isolation and culture techniques used for these bacteria are relatively simple and many strains can be obtained with minimum effort. Moreover, because studies of marine bacteria have not been extensive, the strains isolated will likely be ncw to natural products research. This approach,
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however, neglects the possibility that other diverse taxa produce novel secon dary metabolites. Expanding natural products studies to i nclude diverse bacterial groups (e.g. anaerobes) requires specific microbiological expertise and presents the oppor tu ni ty for collaborations between natural products chemists and microbiolo gists familiar with these methodologies. The challenges of establishing a microbial screening program, especially one that includes diverse bacterial taxa, are well known to the pharmaceutical i ndustry,and the approaches used to meet these challenges have been described (e.g. 12,19,29,148). Although a correlation between taxonomic and biosynthetic diversity has yet to be
defined for marine microorganisms, the inclusion of diverse bacterial taxa in
natural products research still represents a rational approach to novel metabo lite discovery.
The Distribution of Bacteria in the Sea
The factors controlling the distribution of bacteria in the sea are complex and vastly different from those experienced by terrestrial bacteria. The most pro
found of these differences may well be the aqueous milieu itself, which pro
vides an effective medium for ba cteri al

typically contains 104 to 106 bacteria ml-1 greater variety
dispersa l.
Considering that seawater
(4),
and ocean currents can transport
a far of ha bitats and environmental conditions than terrestrial bacte ria. The ability of marine bacteria to surviv e in and utilize the resources found in these various habitats will ultimately have a significant effect upc;m their distribution in the sea. The habitats that marine bacteria encounter include surfaces, both animate and inanimate. In contrast to terrestrial systems, marine surfaces are c onti nually bathed by an assemblage of bacteria. The suitability of these various surfaces for colonization may significantly affect bacterial distributions in marine habi
tats. Although the numbers and types of bacteria colonizing marine surfaces have not been adequately documented, the colonization process and the factors
lhese organisms considerable distances, marine bacteria may encounter
affecting colonization have been discussed in detail (24, 41,101, 117, 187). From the perspective of natural product discovery, a general understanding of the distribution of marine bacteria and the factors affecting these distribu tions is beneficial. If a specific taxonomic group of bacteria is chosen for chemical study, i nformation describing the distribution of that group will facilitate the collection of appropriate samples. In general,these samples may inc lud e seawater, sediments, and both animate and inanimate objects [S ie burt h (161) has reviewed the associations of bacteria with marine samples]. Consid ering that the distri bution of bacteria is not uniform throughout the marine environment, qualitative descriptions of these distributions can be used to maximize the number of diverse bacterial taxa obtained in culture.
SECONDARY METABOLlTES FROM MARINE BACTERIA
565
In the course of isolating marine bacteria for chemical evaluation, we have surveyed the distributions of one chemically significant group, the actinomy cetes. The best marine source of actinomycetes identified thus far are sedi ments, from which their isolation is well documented (8, 5 3, 63, 82, 144, 1 45,
1 72, 185, 1 92). The relative numbers of actinomycetes in marine sediments

are low, however, and selective isolation methods (e.g. 34, 54, 69, 99, 1 12,
1 26), which include heat and antibiotic treatment, are useful. Following iso
lation, methods suitable for the culture and chemical evaluation of marine actinomycetes include those used historically by the pharmaceutical industry for soil actinomycetes, with the most common difference being the incorpo ration of seawater or specific marine nutrients into the growth medium (e.g.
1 36).
The value of studying actinomycete distributions in sediments is demon strated by the observation that their numbers can vary depending upon the depth from which samples are collected ( 1 91, 1 92). For example, in a study of near-shore marine sediments, streptomycetes were encountered infrequently past the sublittoral zone, while actinoplanetes were isolated in greater numbers as the distance from shore increased (82). This information is not only useful when targeting specific taxa for isolation but can be used to assess the indige nous nature of actinomycete isolates-an important consideration, as the origin and activity of actinomycetes isolated from marine sources has been questioned (53, 56, 137). These questions are valid given that actinomycetes are more abundant in terrestrial soils than in marine sediments (55), show varying degrees of salt tolerance (98, 1 34, 1 37, 1 77), and produce spores that undoubt edly wash in large numbers from shore into the sea where they may remain viable for an undetermined period of time. Although questions about the indigenous marine nature of actinomycetes are difficult to address experimentally, some progress has been made in this regard. For example, we have observed that the degree to which marine-derived actinomycetes require saline conditions for growth varies depending upon the taxonomic group studied (82). In this example, virtually all sediment-derived actinoplanetes required seawater for growth and were found in greater numbers as distances from shore increased. These observations suggest that the acti noplanetes were highly adapted to the marine environment and represent obligate marine bacteria. On the other hand, the number of streptomycetes decreased as distances from shore increased. These bacteria could grow in a nonmarine medium, which indicates that the streptomycetes are facultatively marine and possibly of terrestrial origin. Regardless of origin or taxonomic affiliation, all of the actinomycetes isolated grew luxuriantly in a seawater based medium and therefore have the potential to not only be metabolically active in the marine environment but to produce unique metabolites when provided with specific marine growth factors.
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The Associations of Bacteria with Plants and Invertebrates

The distribution of bacteria on living marine surfaces can reveal the extent to which these organisms are consistently associated with specific plants and animals. Documenting the specificity of these associations is an integral (but not in itself conclusive) part of defining bacterial symbioses. In the quest for new natural products, these associations are particularly significant, because the potential role of bacterial symbionts in the production of metabolites previously ascribed to other organisms is open to debate (see section on bacterial symbioses) (see
37, 39). Without evidence for the consistent associa
tion of bacteria with a given plant or invertebrate, one cannot distinguish symbionts from transient associates, and the role of symbiotic bacteria in metabolite production becomes obscured. Despite increasing documentation of the coexistence between bacteria and marine invertebrates (see lacking
160), conclusive evidence of symbioses is often (14). Among the invertebrates frequently collected for natural product (194)]. These studies showed that sponge
study, sponge-bacterial associations are probably the most thoroughly de scribed [see review by Wilkinson associated bacteria can be distinct from bacteria in the surrounding seawater
(156, 193, 195). Also, phenotypically similar bacteria were isolated from 9 of 1 0 sponge species collected from two distinct geographical regions (195).
These results suggest that the bacterial communities associated with sponges may be highly specific. However, this specificity may not apply to all sponges (e.g.
111).
In the case of coelenterates, another group of organisms typically collected for chemical study, the mucus of individuals within the Orders Zoanthidae and Gorgonacea can harbor dense bacterial communities
(33, 155, 183). Despite
these observations, little is known about how the communities vary within or between species. In addition, bacteria associated with algae can be distinct from bacteria in the surrounding seawater (13,
84, 102, 106), but the specificity
of these associations is not clear. Given the limited number of bacterial asso ciations described relative to the number of diverse marine organisms available for study, this subject remains a relatively unexplored area for future research. Although much remains unknown about bacterial associations with marine plants and invertebrates, many highly specific associations have been docu mented. These examples include the associations of bioluminescent Vibrio fischeri with the light organ of cephalopods (190), dinitrogen-fixing bacteria with both the gastrointestinal tract of sea urchins (57) and the gland of Deshayes in shipworms (188), subcuticular bacteria with echinoderms (66, 105), and the spirochete Cristispira sp. with the crystalline style of molluscs (96). The broad spectrum of these associations suggests that specific bacterial invertebrate symbioses are common in the sea. Future studies may even show
567
that the majority of marine organisms have specific bacterial populations associated with their external surfaces and internal tissues. A recent observation in our laboratory indicates that unusual and previously undocumented bacterial associations occur with marine algae. During a study of bacteria in the coral reef environments of Belize, Central America, we found that a large percentage of the culturable bacteria associated with algal surfaces were gram positive. More specifically, 30% of the bacteria associated with the green alga Halimeda sp. and the brown alga Lobophora variegata were gram positive (83). This observation contradicts the early belief that 95% of marine bacteria are gram negative (201). Although gram-positive bacteria have been reported in greater abundance in sediments (118), the relative abundance of these bacteria in most marine habitats has not been adequately assessed. We have included unicellular, gram-positive marine isolates in our studies of bacterial secondary metabolite production. Of approximately 500 strains screened thus far, promising insecticidal activity, enzyme production, cyto toxicity , and at least one novel molecule (178) have been detected. Although marine gram-positive bacteria do not appear to rival the actinomycetes in their ability to produce large quantities of structurally complex secondary metabo lites, additional studies of these bacteria are clearly warranted. As investiga tions into the distribution of gram-positive and other bacteria on the surfaces of marine plants and invertebrates continue, previously undocumented asso ciations may be revealed. These associations may reflect bacterial survival strategies that include the production of unusual secondary metabolites, as well as provide important information relating to the bacterial production of meta bolites previously ascribed to other sources.
The Role of Symbiotic Bacteria in Secondary Metabolite Production
Historically, the search for marine natural products has centered around chemi cal studies of marine plants and soft-bodied invertebrates. Considering that virtually all organisms collected for chemical study include associated micro organisms, questions about the true biosynthetic origin of molecules isolated from plants and invertebrates must be addressed. In some cases, circumstantial evidence leads to the suspicion that metabolites are of bacterial origin. This evidence can include extremely low (e.g. lO-6% by weight) and often variable metabolite yields, the isolation of the same metabolite from diverse inverte brate taxa, consistent microbial associates in the producing organism, and the similarity of the molecule in question to a previously described microbial product. Addressing questions of biosynthetic origin are particularly appropri ate when one considers that in certain marine invertebrates. e.g. sponges, associated bacteria can account for up to 40% of the cellular volume (194). Researchers investigating the biosynthetic origins of marine metabolites
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have taken several experimental approaches to address this question. The most common approach has been the isolation and culture of bacteria from the producing organism followed by chemical analysis of the cultures for the metabolite of interest. In most cases, these efforts have been unsuccessful, which is not surprising when one considers the likelihood of obtaining the producing strain in culture and providing this strain with the culture conditions necessary for metabolite production. If the bacterial production of a metabolite cannot be demonstrated in culture, the documentation of a specific bacterial population associated w ith the producing organism, in an abundance that can account for the concentration of the metabolite in the host, can provide impor tant data in support of bacterial b iosynthesis. Probably the most notable example of the bacterial production of a meta bolite originally ascribed to another organism involves the potent marine neurotoxin, tetrototoxin (TTX), best known for the illness and death it can inflict following the consumption of tainted, raw pufferfish (Fugu spp.). In vestigations into the origin of TTX (114) revealed that TTX is produced as a fermentation product of both a Vibrio sp. isolated from the intestines of a xanthid crab, Atergatis floridus (125), and a Pseudomonas sp. isolated from a red calcareous alga,Jania sp. (199). More recent studies have shown that TTX is produced by a variety of taxonomically diverse marine bacteria (31, 1 64, 200), including actinomycetes ( 30). This information has led to the proposal that bacteria are the sole origin of TTX, which may subsequently accumulate in invertebrates through the food web ( 31). In addition to TTX, bacteria have been found to produce at least two other marine toxins. One example comes from the edib le mollusc Babylonia japon ica (94), the consumption of which caused at least one outbreak of food poisoning in Japan. B. japonica toxicity was traced to the presence of neosu rugatoxin and prosurugatoxin, both of which were isolated from the digestive gland of the toxic mollusc. Microbiological studies revealed that a coryneform bacterium,also isolated from the digestive gland of B. japonica, could produce these toxins. A third example of bacterial toxin production is saxitoxin (STX), one of the major causative agents in paralytic shellfish poisoning. STX has been isolated from dinoflagellates, e.g. Protogonyaulax tamarensis (15 8), which were presumed to be the source of shellfish toxicity. However,shellfish toxicity is not correlated with the presence of P. tamarensis (129). Recent studies revealed that a Moraxella sp. isolated from P. tamarensis produces STX (89-91 ) and may be the u ltimate origin of this metabolite. In at least two additional studies, the bacterial production of metabolites previously ascribed to nonbacterial sources has been reported. The first (165) demonstrated that a Micrococcus sp., isolated from the sponge Tedania ignis, produced a series of diketopiperazines previously reported from the sponge. In the second study (35), brominated diphenyl ethers originally isolated from
569
the sponge Dysidea sp. were obtained as fermentation products from two strains of Vibrio sp. reported to b e symbionts of Dysidea. Despite such evi dence of bacterial origin, critical reading of these papers leaves room for speculation about the true biosynthetic origin of the sponge metabolites (37, 39). Other attempts to determine the biosynthetic origin of marine metabolites include the physical separation of bacteria from host tissues,and the subsequent examination of these cells for the presence of the metabolite in question. The most successful demonstration of this technique involved the use of flow cytometry to separate cyanobacterial symbionts (Oscillatoria spongeliae) from the sponge Dysidea herbacea (181). Through analysis of the purified cyano bacteria, these authors provided the first evidence for the localization, in prokaryotic symbiont cells, of a secondary metabolite previously ascribed to an invertebrate host. Other efforts to determine the cellular location of bacterial metabolites include the use of molecular probes; however, to the best of our knowledge these methods have not yet met with success. Bacterial associations with p lants and animals pose challenging questions in the field of marine natural products chemistry. Currently, the questions of greatest interest involve the role of bacteria in the biosy nthesis of metabolites previously ascribed to other organisms. Part of this interest is purely ecological, but this subject becomes especially important when the metabolite in question shows promising biomedical applications. One such case is bryostatin 1, the anticancer agent isolated from the bryozoan Bugula neritina. In the case of bryostatin 1, the evidence for bacterial origin includes the low yields (-10-6% by weight) in which the metabolite is produced and the presence of distinct populations of bacterial symbionts in the bryozoan tissues. When one considers that B. neritina is not available from nature in quantities that can effectively facilitate biomedical development, the possibility of producing bryostatin I by fermentation becomes a high priority.
Effects of Marine MetaboLites upon the Distribution of Bacteria
Because of the lack of experimental evidence, we can only speculate about the effects of plant and invertebrate metabolites upon the distribution of bac teria in the sea. These effects may be expressed in several ways including the deterrence of u ndesirable bacteria or the attraction of bacteria that represent favorable epibionts. A lthough laboratory-based assays have indicated that bac teria can respond to marine metabolites (e.g. 10,20), little ecologically relevant data are available to suggest these substances have an effect upon surface colonizing bacteria (see 21 for a review of ecological aspects affecting micro bial chemotactic behavior). Early evidence suggesting the involvement of marine metabolites in the control of epibacterial distributions came from studies of marine algae. In two
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separate reports, investigators proposed that antibiotic algal phenols were responsible for reduced numbers of surface bacteria and fouling organisms on the marine algae Sargassum natans (162) and Ascophyllum nodosum (25). Subsequently , a correlation between reduced levels of epibiosis and the pro duction of antimicrobial substances by marine organisms was presented as evidence for the ecological role of these metabolites in antifoulIng (e.g. 1,27, 115, 174, 182, 186). Unfortunately,the degree to which antimicrobial activities translate to in situ effects upon potential surface-colonizing bacteria has not been adequately demonstrated. A lthough the in situ microbiological effects of marine natural products remain largely undocumented, two findings suggest they may influence the distribution of bacteria on living marine surfaces. One example comes from the study of two colonial ascidians that possessed remarkably different num bers of epibiotic bacteria (184). Laboratory settlement assays comparing these ascidians demonstrated that extracts of the sparsely colonized Cystodytes 10batus were highly deterrent to bacterial attachment while extracts of the densely colonized Polyclinum planum significantly enhanced the attachment process. These results suggest that the mediation of bacterial attachment by host metabolites may be an important factor in the control of bacteria upon living marine surfaces. A second example demonstrating the effects of secondary metabolites upon the distribution of microorganisms involves the inhibition of pathogenic fungi by bacteria associated with the embryos of two marine crustaceans, Palaemon macrodactylus and Homarus americanus. In separate studies,researchers ob served that embryos of both P. macrodactylus and H. americanus were covered by dense assemblages of what appeared to be largely unibacterial populations (50,51). Bacteria consistently isolated from the embryos of P. macrodactylus and H. americanus produced the previously described metabolites isatin and tyrosol, respectively, both of which show antifungal effects toward the crus tacean pathogen,Lagenidium callinectes. Further experiments with P. macro dactylus revealed that bacteria-free embryos rapidly succumb to fungal infection whereas similar embryos re-inoculated with bacteria survived. These results led to the conclusion that symbiotic bacteria chemically defend P. macrodactylus embryos against fungal infection. Bacterial associations of this nature add a new level of complexity to the mechanisms by which secondary metabolites influence the distributions and specific associations of marine microorganisms.
The Adaptations of Bacteria to Diverse Marine Habitats
Their metabolic capacity allows prokaryotes to survive virtually across the spectrum of environmental conditions found on this planet. Assuming that metabolic and biosynthetic di versity are correlated, the adaptations of bacteria
571
to diverse marine habitats provide seemingly limitless evolutionary opportu nities for the production of unique secondary metabolites. Although bacteria adapted to diverse marine environments have apparently received little atten tion in the search for novel secondary metabolites, an important industrial application has corne from the study of one such group, i.e. the thermophilic archaea (Archaebacteria) associated with deep-sea hydrothermal vents (SO). These bacteria produce thermostable DNA polymerase enzymes, which have been isolated, cloned, and marketed for biotechnological applications (7S). Other evidence of bacterial adaptations to extreme marine environments in cludes the recognition that a gene (ompH) from a gram-negative bacterium isolated from the depths of the Sulu Sea is expressed only at increased hydro
static pressure (9). This discovery suggests the existence of a genetic program
that couples pressure sensing to protein synthesis. Bacterial Culturability Maintaining bacteria in culture is requisite to the effective study of secondary metabolite production, because adequate quantities of b acterial biomass can seldom be obtained from samples collected in the field. Unfortunately, the majority of bacteria observed in certain marine samples do not form colonies on the nutrient-rich agar traditionally used for the isolation of marine bacteria (81,92). In fact, microscopic counts performed directly on seawater samples, usually accomplished with florochrome stains (e.g. 64, 146), typically yield bacterial counts three orders of magnitude higher than those obtained with plating techniques (e.g. 163). These results have led to the generally accepted conclusion that the majority of marine bacteria are unculturable,a conclusion that raises serious concerns for researchers interested in maximizing the num ber of diverse isolates screened for unique secondary metabolites. Several topics deserve closer scrutiny in the discussion of bacterial cul turability. For one, most studies have focused upon the b acteria in seawater samples collected far from shore. This may be important considering that the organic content of open-ocean seawater is generally low (116),and that seawa ter bacteria have evolved survival strategies (154), including responses to starvation (97, 127), that may dramatically reduce their ability to form colonies on nutrient-rich agar (e. g. Difco 2216). Furthermore,the discrepancy between viable and direct counts, for some seawater samples, may result from low plating efficiencies, i.e. only a few viable individuals in a population are capable of forming colonies on agar media (147). It should also be noted that the percentage of culturable b acteria calculated for marine samples can be higher than the 0.1% typically reported for seawater. For example, 3.4 to 11% of the bacteria observed in association with the sclerosponge Ceraloporella nicholsoni were culturable (156). If living marine surface s provide nutrient-rich environments, a relatively large percentage of
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the bacteria associated with these surfaces may form colonies when inoculated onto nutrient-rich growth media. To the best of our knowledge, the culturablil ity of bacteria associated with nutrient-rich marine habitats has not been ad dressed in detail. One habitat that would be ideal for such studies is the marine invertebrate digestive system, which can harbor extensive bacterial communi ties
(61).
Another concept that must be kept in mind in discussions of bacterial culturability is the inherent selectivity of all isolation methods. Because only a limited group of bacteria will grow when any one isolation method is used, it is not surprising that large numbers of bacteria appear to be unculturable. To determine the percentage of culturable bacteria in any community, one would have to attempt multiple isolation methods and determine the sum of all unique strains resulting from these methods. This is not feasible; hence the percentage of culturable bacteria reported for any one sample must be consid ered in the context of the isolation methods used. We can also ask questions about the composition of the nonculturable component of the bacterial community. Do these bacteria represent noncultur able strains of previously described species, or do they represent new species or genera? Recent analyses of small subunit
( 1 6S ) bacterial rRNA sequences
have shed light on this subject. For example, it has been shown that bacte rioplankton communities consist of broadly diverse bacterial assemblages within which undescribed phylogenetic groups are common assess the culturability of these bacteria. Apparently, for taxonomically diverse marine bacteria to be effectively incorporated into the search for new microbial products, new culture methods need to be developed, particularly methods that take into account the environ mental parameters associated with the habitats sampled. These methods may include the use of marine-derived nutrients, which have been shown to induce the production of novel metabolites in at least one report
(43). As we be
come aware of novel bacterial populations, focused efforts can be made to
( 1 36). In this study,
the addition of a pulverized brown alga, kobu cha (Laminaria sp.), to the growth medium induced the production of a new antibiotic, SS-228 Y. The development of marine-derived nutrients may improve our ability to culture marine bacteria and present additional opportunities for the discovery of novel metabolites.
The Distinction Between Marine and Terrestrial Bacteria

Establishing the existence of specific marine bacteria was fundamental to the development of marine microbiology as a scientific discipline. Early studies led to the proposal that marinc bactcria can bc distinguishcd from terrestrial strains based upon the requirement of seawater for growth
(202). Subsequent
studies documented a readily detectable sodium requirement for growth, which
573
is rarely observed for terrestrial strains (110). Although marine bacteria lack an official definition, this demonstration of a specific sodium requirement for growth has become widely accepted as a method to distinguish marine from most terrestrial bacteria [see reviews by MacLeod (l08, 109)]. More recently, the physiological role of sodium was investigated in greater detaiL For many marine bacteria,the transport of various solutes into the cell is sodium dependent (11, 175, 196). The energy required for this transport i s gen erated by a respiration-dependent sodium pump (175). This pump extrudes sodium ions,generating a transmembrane sodium gradient with potential en ergy that can be utilized to perform various cellular functions including the generation of ATP. Oh et al (130) have shown that the sodium requirement in marine bacteria is correlated with the possession of respiration-dependent sodium efflux,and these authors have suggested that electron transport-driven sodium efflux can b e regarded as a criterion for the definition of marine bacteria. Although the physiological distinctions between marine and terrestrial bac teria are clearly important, the rationale for restricting marine bacteria to only those strains that meet a predefined set of requirements is questionable. For example, in studi es conducted off the coast of Belize, we observed that more than 80% of the gram-positive bacteria isolated from a variety of sources required seawater for growth (83). In addition, all seawater-requiring strains tested had a demonstrable requirement of sodium for growth. As a result, these bacteria can be defined as marine based upon traditional proposals (110, 202). Also based on these proposals,bacteria that did not require seawater or sodium for growth,but w ere capabl e of growth in seawater-based medium,could be labeled as halotolerant terrestrial strains. But,should w e reach this conclusion a priori, and as a result eliminate non-seawater- and/or non-sodium-requiring bacteria from discussions of marine bacteria? To take this question one step further, we can envision a number of marine niches, e.g. those inhabited by intracellular symbionts, in which bacteria may never encounter seawater. If these bacteria do not have a demonstrable sodium requirement, aren't they nonethel ess marine? We believe that by restricting marine bacteria to include only those strains that meet a predefined set of requirements we run the risk of overlooking certain groups that may be equally well adapted to survive on land or in the sea, as w ell as indigenous marine bacteria that simply do not meet the set of requirements chosen. Furthermore,the requirement of seawater for the growth of marine bacteria appears to vary between taxa. In the case of gram-positive bacteria, none of the cocci tested in at least four separate studies required seawater for growth (83, 93, 100, 197). Also,as currently described the genus Planococcus, which is recognized as a marine genus, does not require seawater for growth (23, 88, 128). MacLeod (109) makes an interesting observation regarding the gram-
574
JENSEN & FENICAL
positive coccus that
Gaffkya homari
(now
Aerococcus viridans),
the pathogenic
bacterium that causes septicemia in lobsters. Citing unpublished data, he states
G. homari
does not require seawater for growth and that at least among
the gram-positive species in the sea, some representatives of the indigenous flora require neither seawater nor sodium for growth. With the exception of
Marinococcus spp., Salinococcus spp., Sporosarcina halophila, and Micrococcus halobius (23, 60, 1 1 3), which grow optimally in 20% salt solutions and are typically isolated from solar salterns,
salted food products, and other hypersaline environments, there appears to be a trend in which gram-positive cocci isolated from the sea do not require seawater or sodium for growth. This subject certainly warrants further inves tigation. In our opinion, to make a distinction between marine and terrestrial bacteria based upon sodium requirements is not as important for natural product dis covery as the demonstration that bacteria isolated from the sea perform unique biosyntheses. The best example of unique marine biosyntheses may well be the bacterial production of halogenated antibiotics (e.g.
the moderately halophilic cocci, e.g.
2, 1 8, 1 40). If the
production of these and other metabolites that possess features seldom encoun tered outside the realm of the ocean is restricted to a specific set of marine conditions, then chemical studies of bacteria grown in these conditions are warranted.
BIOLOGICAL ACTIVITIES AND NOVEL ORGANIC SUBSTANCES

The ability of marine bacteria to produce antibacterial substances was docu mented more than coworkers
45 years ago ( 1 53). Nearly 20 years later, Burkholder and ( 1 8) purified the first antibiotic from a marine bacterium, and later that same year, Lovell ( 107) reported the structure of this substance, a highly
brominated pyrrole. In the years that followed these early discoveries, the number of novel organic molecules reported from marine bacteria has grown to include nearly
30 substances (Table 1 ). These substances represent diverse
classes of chemical structures and provide evidence for the untapped biosyn thetic potential of marine bacteria. Although the total number of unique mole cules reported to date remains low, approximately one half of these were described after
1 990, reflecting a growing interest in this area of research. 1
The biological activities reported for the molecules described in Table
include not only antibiotic, but also anticancer and antiviral activities. To some extent, these activities are a reflection of bioassay accessibility and the scien tific objectives of the research group responsible for the discovery. Although the rapid screening of bacterial extracts is essential to biomedically relevant
575
discovery (because of low hit rates) and to effective bioassay-guided chemical isolation, these bioassays can become burdensome for small research groups. Maximizing the potential for drug discovcry through in-house testing is also difficult for small research groups considering the many new and sophisticated target-directed screens developed in recent years. For these reasons, industrial and academic collaborations offer an effectiv e avenue by which to obtain broad-spectrum screening of bacterial metabolites. As efforts to screen marine microbial products increase,additional biomedically and industrially important activities will c ertainly be discovered. We mentioned that novel secondary metabolites have been isolated from a limited taxonomic group of marine bacteria. This may be misleading as othcr genera, including Moraxella (52) and Alcaligenes ( 1 6) produce biologically active substances that have not been fully characterized. In addition,an anti tumor polysaccharide has b een isolated from a Flavobacterium sp. ( 1 80), and antibiotic activity was associated with a purple photosynthetic bacterium, Chromatium purpuratum ( 1 7). However, to the best of our knowledge many taxa have not been studied in detail for the production of biologically active secondary metabolites. One genus that is conspicuously absent from the marine natural products literature is Bacillus, even though isolates are readily obtained from marine sediments ( 1 6 1 ) and unusual secondary metabolites have been reported from terrestrial strains ( 1 2). Other than the recent isolation in our laboratory of a previously undescribed cyclic depsi-peptide ( 1 78), to the best of our knowl edge,only one other substance-an enzyme-has been reported from a marine Bacillus sp. ( 1 33). The archaea were also absent from the marine natural products literature prior to the recent isolation of new cyclic polysulfides from strains of anaerobically cultured Thermococcus spp. ( 1 52). These thermophilic, sulfur-metabolizing archaea were collected near marine hydrothermal systems and the substances produced demonstrated both antifungal and anthelmintic acti vities. In addition to the cases already mentioned,the history of biological activities reported for marine bacteria is long. For example, after the survey of antibiotic activity by Rosenfeld & Zobell ( 153) came demonstrations that marine bacteria possess antiviral (52, 1 76), anti-algal (62), anticancer ( 1 80), and antimicrobial activities (6, 7, 1 5-17, 32, 44-48, 56, 95, 103, 104, 1 1 9). Investigators have also demonstrated that marine bacteria produce bacteriocins (68) and enzyme inhibitors (70-75, 1 7 1). In some cases, these activities are due to proteins or other large molecules for which structural elucidation cannot be performed using standard spectral techniques. In other cases, attempts to isolate and characterize the active principles were never made. The extent of these activi ties, however, supports further investigation of secondary metabolites from marine bacteria.
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JENSEN & FENICAL
CONCLUDING REMARKS Today, marine bacteria are being recognized as an important resource for microbial products. The growing interest in this resource is adequately dem onstrated by the number of novel metabolites recently reported from marine bacteria and the number of research laboratories throughout the world now working in this field. For the chemical potential of marine bacteria to be effectively assessed, however, continued basic research in marine microbiol ogy is essential. This research is needed because we do not yet have a clear understanding of the types of marine bacteria available for chemical study, the niches they occupy, and the mechanisms by which they are adapted to the marine environment. The focus of our research in marine microbiology is to isolate novel secon dary metabolites from marine bacteria and to ask ecological questions about both the producing strains and the roles of the metabolites produced. In the course of this work, it seems appropriate to consider our results in terms of traditional concepts in marine microbiology. For example, should we distin
guish marine from terrestrial bacteria based upon sodium ion requirements, or
for that matter, any specific physiological criteria? Although the elucidation of specific ion requirements and the function of these ions in bacterial growth is clearly important from a physiological perspective, these adaptations may prove to be less important, in an ecological context, than the demonstration that bacteria are metabolically active in the marine environment. Certainly in terms of natural product discovery, a marine isolate's ability to produce sec
ondary metabolites only when provided with conditions specific to the marine
environment is a primary consideration. Another general concept in marine microbiology that can be addressed in greater detail, especially in light of available modem techniques, is the number and activity of gram-positive bacteria in the sea. The relative numbers of gram-positive bacteria in some marine habitats may be greater than previously believed. For example, a recent study demonstrated that 9 of 6 1 clones, ob tained from seawater samples and examined by small subunit ( 16S) rRNA sequence analysis, clustered within a group of gram-positive bacteria (43). Although this study was not intended to be quantitative, it suggests that gram positive bacteria may represent a numerically significant component of the total viable as well as the culturable (83) bacterial community. Clearly, addi tional descriptions of gram-positive bacteria are needed to better clarify their numbers and activities in the sea. To date, only the most preliminary investigations have been made in the search for novel metabolites from marine bacteria. From an academic perspec tive, many ecological questions fundamental to this field remain unanswered. These q uestions include the effects of plant and invertebrate metabolites up on
577
the distribution of marine bacteria and the roles of these metabolites in chemi cal defense against microbial pathogens. Other important questions address the role of symbiotic bacteria in the production of metabolites previously ascribed to nonbacterial sources. Without continued ecological studies of ma rine bacteria, their potential for the production of novel secondary metabolites may never be fully realized.
ACKNOWLEDGMENTS
We thank Professors Ralph Mitchell and Farooq Azam for their critical evalu ation of this manuscript. In addition, we thank Carole Bewley for helpful editorial comments.
Any Annual Review chapter, as well as any article cited in an Annual Review chapter, may be purchased from the Annual Reviews Preprints and Reprints service. 1 .800.347.8007; 415.259.5017; email: arpr@eiass.org
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STRATEGIES FOR THE DISCOVERY OF SECONDARY METABOLITES FROM MARINE

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1977 to date (36, and previous reviews cited therein).