Marine Chemical Ecology - Chapter 18 (end) doc

567

Metabolites of Free-Living,
Commensal, and Symbiotic
Benthic Marine
Microorganisms

Valerie S. Bernan

CONTENTS

I. Introduction 567
II. Marine Sediments 568
A. Shallow Marine Sediments 568
B. Deep-Sea Marine Sediments 574
III. Commensal Marine Microorganisms 577
IV. Symbiotic Marine Microorganisms 583
V. Summary 589
References 589

I. INTRODUCTION

This chapter describes the natural product chemistry that has been identified or associated with
marine microorganisms from the benthos and focuses on the marine eubacteria. Most of the
compounds described in this chapter resulted from a detailed search of the literature for micro-
bially derived natural products. This approach is biased in many respects, since it only describes
bioactive compounds and small molecules. However, due to the growing demand for new thera-
peutic agents, the discovery of new chemical entities is being driven by these efforts.

1

While
comparatively little research has been directed toward the study of natural products from marine
microorganisms, the results to date have been encouraging.

2

Data from these investigations
demonstrate complex chemical interactions between marine bacteria and their hosts,

2

including
systems of signaling and territorial marking. It is estimated that less than 1% of potentially useful
chemicals have been discovered from the marine environment, with microbial products represent-
ing 1% of that total number.

1

Compounds isolated from marine microorganisms have demonstrated
antibiotic, anticancer, anti-inflammatory, and other pharmacological activites.

2,3,4

Hopefully a
greater understanding of microbial metabolic diversity, coupled with ecological information, will
yield a greater understanding of the complexity of marine environments. This chapter reviews
metabolites of microorganisms that occur in sediments, as well as those in commensal associations
and symbiotic relationships.
18

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II. MARINE SEDIMENTS

Marine sediments are composed of organic debris resulting from ongoing, seasonal, or catastrophic
die-offs of macrobiotic and microbial populations.

2

This sedimenting debris provides energy for
the benthic organisms on the sea floor. Organic mineral aggregates that are rich in carbohydrates
are produced by deposit-feeding animals such as bivalves, which rework the sediments as they
feed. This extensive reworking of the sediments makes them unstable so that the organic aggregates
are resuspended by the tidal flow to produce turbid estuarine waters which are important in
recycling. Consumption of resuspended organic debris and phytoplankton by bivalve molluscs and
other benthic invertebrates produces rich deposits of feces and pseudofeces that coat mineral
fragments. These mineral fragments yield additional organic mineral aggregates, which in turn are
heavily colonized by microorganisms. Other sources of organic matter include those derived from
cordgrass and from beds of seagrasses, such as eelgrass and turtle grass as well as mangroves. As
these plants decompose, they are reworked into smaller fragments by deposit feeders, which can
support increased populations of microbiota.
Within interstitial habitats of sandy beaches, particles are trapped in the upper 5-cm surface
layer and give rise to a bacterial–protozoan community.

5

Below this level, a bacterial flora attached
to sand grains removes some of the dissolved organic carbon while supporting a meiofauna
community comprised of nematods and copepods. The biotic community of these intertidal sandflats
is supplemented by the production of organic matter via benthic diatoms which migrate vertically
with the tides.
Since terrestrial actinomycetes have been such prolific producers of bioactive molecules, it was
natural to investigate marine species of the same group. Therefore, it was no surprise that actino-
mycetes isolated from the marine sediments have proven to be one of the most prolific sources of
bioactive secondary compounds.

6,7

Their distribution in sediments varies depending on the depth
from which the sample was collected. In several studies,

Streptomyces

predominated in near-shore
marine sediments, but decreased dramatically past the sublittoral zone. In contrast, actinoplanetes
are found in greater numbers as the distance from the shoreline increases.

A. S

HALLOW

M

ARINE

S

EDIMENTS

The study of the metabolites produced by marine actinomycetes was pioneered by researchers at
the Institute of Microbial Chemistry in Tokyo in the early 1970s. One of the first compounds
described by Okami was a benzanthraquinone antibiotic isolated from the actinomycete

Chainia
purpurgensa

SS-228 collected in mud samples from Sagami Bay, Japan.

8

This antibiotic selectively
inhibited Gram-positive bacteria and was active against Ehrlich ascites tumor cells in mice. It also
produced a hypotensive effect in mice, probably due to its inhibition of dopamine hydroxylase in
the pathway of epinephrine biosynthesis. Most interestingly, this bacterium only produced the
antibiotic when it was fermented in diluted yeast extract containing “Kobu Cha” (the brown seaweed

Laminaria

) and with the addition of 3% NaCl. This observation demonstrated that marine micro-
organisms have nutritional requirements corresponding to nutrients in their natural habitats. This
study was also one of the first to report that bacteria from the order Actinomycetales could be
isolated from the marine environment. Actinomycetes were originally thought to have entered the
marine environment via rivers or runoff or to have existed as spores of terrestrial species. However,
work by Moran et al.,

9

using a 16S rRNA genus-specific probe, demonstrated that

Streptomyces

occurred as indigenous populations, and that populations increased in relative abundance in response
to the availability of certain nutrients. Greater abundances of culturable streptomycetes found in
coastal environments vs. deepwater marine systems may be attributed to higher amounts of organic
detritus, much of which is derived from vascular plants concentrated in shallow marine systems.
Unlike the terrestrial actinomycetes, marine actinomycetes have been shown to produce mac-
rolides only rarely. One example of this class of compounds is the aplasmomycins A–C

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Metabolites of Free-Living, Commensal, and Symbiotic Benthic Marine Microorganisms

569

(Structure 18.1a–c). These compounds were isolated from the fermentation of

Streptomyces griseus

SS-20 collected from shallow mud in Sagami Bay, Japan. The antibiotic was only produced under
low-nutrient media containing “Kobu Cha” and 3% NaCl. The aplasmomycins are inhibitory against
Gram-positive bacteria and the bacteria

Corynebacterium smegmatis

. More importantly, aplasmo-
mycin is an effective antimalarial agent in

Plasmodium berghei

-infected mice. X-ray crystallogra-
phy revealed that the active compound contained a symmetric ring in which boron was coordinated
in the center with a crown ether-like structure.

10

Fenical’s group

11

from Scripps Institute of Ocean-
ography also isolated a new member of a rare class of macrolides, maduralide (Structure 18.2).
Maduralide was isolated from the fermentation of a Maduramycete isolated from shallow sediments
from Bodega Bay, California. This compound is a member of a rare 24-membered ring lactone
group represented by rectilavendomycin.
Among the alkaloids, the most unusual example is an acaricidal (lethal to arachnids) monot-
erpene derivative, altemicidin (Structure 18.3). This novel alkaloid was purified from a marine strain
of

Streptomyces



sioyaensis

SA-1758 isolated from marine sediments collected from the northern
part of Japan. It yielded potent antitumor activity

in vitro

against L1210 murine leukemia and IMC
carcinoma cell lines, but was toxic

in vivo

in mice. Altemicidin is a novel sulfur- and nitrogen-
containing microbial metabolite with a monoterpene carbon skeleton.

12

A sediment-derived

Streptomyces

sp. was isolated from Laguna de Terminos from the Gulf of
Mexico.

13

When the culture was fermented in 50% seawater, a new anti-algal anthranilamide
derivative was isolated that was active against several algae including

Chlorella

spp. and

Scenedesmus subspicatus

. This new compound was shown to be an

N

-methyl anthranilamide
derivative of phenylpropionic acid (Structure 18.4). A series of analogs was synthesized and their
anti-algae properties examined. These methyl ester analogs were found to be more active against
algae than the free acids in both agar diffusion and liquid test systems. The compounds did not
exhibit any antimicrobial activity against

Staphylococcus aureus

,

Escherichia coli

, or

Mucor miehei

at concentrations up to 200

µ

g/mL.
Another

Streptomyces

sp. obtained from black anaerobic intertidal sediment collected near
Christchurch, New Zealand was fermented in a saline medium and found to produce modest
antibacterial activity against

Bacillus subtilis

.

14

The isolation of the active compound revealed it to
be an actinoflavoside, a molecule of an unprecedented structure class (Structure 18.5). Actinofla-
voside resembles the common plant-derived flavonoid glycosides, but this compound contains an
O
O
O
O
O
O
O
O
O
O
O
O
B
OR

R'O
-
M
+
18.1a: Aplasmomycin A R = R' = H
18.1b: Aplasmomycin B R = H, R' = Ac
18.1c: Aplasmomycin C R = R' = Ac

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Marine Chemical Ecology

additional alkylation at C-5. Due to the general conclusion that prokaryotes do not produce this
class of compounds, its origin via the flavinoid biosythetic pathway seems questionable.
A marine actinomycete,

Streptomyces virdostaticus

spp. "

litoralis

" was isolated from an inter-
tidal sample collected in Key West, Florida. Fermentation samples exhibited both antibacterial and
DNA-damaging activites. Although activity was observed in a tap-water based medium, the addition
of 2% NaCl increased the biomass by 33% and increased activity four-fold. Purification of the
active materials revealed four related bioxalomycins (Structure 18.6a and b). The

β

species were
found to be the quinone forms of the corresponding

α

components. The

β

components are distin-
guished from the antibiotic naphthyridinomycin by the presence of a second oxazolidine ring in a
region of the molecule analogous to quinocarcin. Bioxalomycin

α

2 was the most potent antibiotic
of the group, showing MIC values between 0.002–0.25

µ

g/mL, and also exhibited excellent

in vitro

activity against neoplastic cell lines. This compound was active

in vivo

in a mouse P388 leukemia
model demonstrating an 80% increase in life survival (ILS). Mechanistic studies have shown that
following metabolic reduction of the quinone, bioxalomycin

α

2 cross-links DNA through alkylation
of guanine residues in the minor groove of DNA.

15

A new series of phenazines has been isolated from a strain of marine-derived

Streptomyces

by
Fenical’s group.

16

A study from the shallow sediments of Bodega Bay, California resulted in the
N
COOH
CH
3
O
18.4: Anthranilamides
N
CONH

2
HN
OH
COOH
SO
2
NH
2
O
CH
3
H
H
18.3: Altemicidin
OH O
O
OO
H
OH
O
O
OH
OH
OCH
3
H
OH
18.2: Maduralide
O
O

O
O
O
OH
HO
NH
HO
H
O
HO
H
18.5: Actinoflavoside

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Metabolites of Free-Living, Commensal, and Symbiotic Benthic Marine Microorganisms

571

isolation of an unknown

Streptomyces

sp. that was found to produce compounds with broad
antibacterial activity. Subsequent fermentation of this isolate in a saltwater based medium produced
four new alkaloid esters of the phenazine class which contained the sugar L-quinovose at the 2

′

and 3

′

positions (Structures 18.7a and b). These compounds were found to exhibit antibacterial
activity against Gram-negative and Gram-positive bacteria.



Another set of phenazine derivatives
was isolated from the fermentation of a

Streptomyces

sp. isolated from a sediment sample collected
in Laguna de Terminos in the Gulf of Mexico.

17

Cultivation and production occurred in enriched
50% seawater medium and produced the novel 5,10-dihydrophencomycin methyl ester and the
known microbial metabolites (2-hydroxyphenyl)acetamide, meanquinone MK9, and phencomycin.
The new 5,10-dihydrophencomycin methyl ester (Structure 18.8) exhibited less antimicrobial activ-
ity than phencomycin, and the dimerization of identical

m

-C

7

N units may explain its origin.
Four new

α

-pyrone-containing metabolites, wailupemycins A–C and 3-epi-5-deoxyenterocin
(Structures 18.9 a–e) were isolated together with the known compounds enterocin and 5-deoxy-
enterocin from the fermentation broth of a new

Streptomyces

sp.

18

The strain was isolated from
the shallow marine sediments collected at Wailupe beach park on the southeast shore of Oahu,
Hawaii. The

α

-pyrone moeity is commonly observed in many antibiotics and toxins. Interestingly,
enterocin and 5-deoxyenterocin, along with the 5-behenate and 5-arachidate esters of enterocin,
has been isolated from a marine ascidian of the genus

Didemnum

. The occurrence of the same
compounds in both prokaryotes and chordates raises the question of whether symbiotic or asso-

ciated microorganisms are responsible for the production of the metabolites isolated from some
marine invertebrates.
In a study of estuarine microorganisms isolated from Torrey Pines, La Jolla, California, Fenical
et al.

19

isolated marinone (Structure 18.10) and its debromo analogue debromomarinone from the
fermentation broth of an unidentified actinomycete. Both compounds possess a common naphtho-
quinone with rare sesquiterpenoid structural components. In addition, marinone possesses a bromine
substituent in the dihydroxybenzene ring, a position typical for bromination in marine metabolites.
These new molecules are among a rare group of bacterial metabolites produced via mixed biosyn-
thesis involving both acetate and terpene pathways. Marinone and debromomarinone exhibit sig-
nificant

in vitro

antibacterial activity against Gram-positive bacteria. More recently, the same group
isolated a different unidentified actinomycete, from a sediment sample collected at 1m depth in
Batiquitos Lagoon, California, that also produced marinone in addition to several cytotoxic metab-
olites related to marinone. One compound, neomarinone (Structure 18.11), is a novel metabolite
possesssing a new sesquiterpene- and polyketide-derived carbon skeleton. The other two derivatives
are isomarinone and methoxydebromomarinone. All three compounds are also derived from a mixed
biosynthetic pathway involving polyketide and terpene pathways. Connection of the sesquiterpenoid
side-chain to the naphthoquinone core occurs on the nonquinone side in neomarinone. The origin
of the sesquiterpenoid side-chain appears complex; it is possibly derived from a cation-induced
methyl migration as observed in the trichothecenes. All three compounds exhibited moderate

in vitro
N

N
N
O
O
OH
OH
CH
3
O
CH
3
R
H
H
OHH
H
18.6a: Bioxalomycin α1, R = H
18.6b: Bioxalomycin α2, R = CH
3

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Marine Chemical Ecology

cytotoxicity in the National Cancer Institute’s 60 cancer cell line panel with a mean IC

50

value
of 10

µ

M.

20

As part of the continuing interest in isolating secondary metabolites from marine estuaries, a
Streptomycete was isolated from a sediment sample collected in Mission Bay, California. When
the culture was fermented under saline culture conditions, it was found to produce a family of
novel cyclic heptapeptides, cyclomarines A, B, and C. While the major metabolite, cyclomarine
A (Structure 18.12), is cytotoxic

in vitro

toward cancer cells, it is more interesting for its significant

in vitro

and

in v

ivo anti-inflammatory properties. The compound displays significant topical anti-
inflammatory activity in the phorbol ester-induced mouse edema assay, showing 92% inhibition
at the standard test dose. Cyclomarine A contains three common and four unusual amino acids.
The four unusual amino acids are

N

-methylhydroxyleucine,

β

-methoxyphenylalanine, 2-amino-
3,5-dimethylhex-4-enoic acid, and

N

-(1,1-dimethyl-2,3-epoxypropyl)-

β

-hydroxytryptophan. The
latter two amino acids have not been previously described, although similar

N

-prenyltryptophan
amino acids have been observed in the ilamycins. The amino acid

β

-methoxyphenylalanine is a
well known synthetic building block, but is a rare constituent of natural products, found in only
N
H

H
N
CO
2
CH
3
CO
2
CH
3
18.8: 5,10-dihydrophencomycin methyl ester
N
N
H
3
COH
O
O
HO
CH
3
OH
R
R
O
N
N
CH
3
OH

O
O
HO
O
R
R
OH
18.7a: R=OH, R=H
18.7b: R=H, R=OH
′
′
′
′

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Metabolites of Free-Living, Commensal, and Symbiotic Benthic Marine Microorganisms

573
O
O
OH
OH
HO
O
CH
3
OO
O

O
OO
OCH
3
HO
O
O
O
O
H
HO
O
O
OCH
3
H
O
O
O
OH
O
CH
3
O
HO
OH
H
H
O
18.9a: Wailupemycin A

18.9b: Wailupemycin B
18.9c: Wailupemycin C
18.9d: Wailupemycin D
H
N
N
H
O
N
O
HN
O
H
N
O
HN
O
N
O
O
HO
N
HO
CH
3
O
O
18.12: Cyclomarin A
O
HO

OH
O
O
18.11: Neomarinone
H
H
OH
HO
Br
O
O
18.10: Marinone

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574

Marine Chemical Ecology

the discokiolides, which are cyclic depsipeptides from the marine sponge

Discodermia kiiensis

.
These amino acids may be products of unusual biosynthetic pathways, and it will be interesting
to elucidate their biosynthesis.

21

Another estuarine

Streptomyces

was isolated from the sandy
sediment collected near San Diego, California in the San Luis Estuary. Cultivation of the

Strep-
tomyces

sp. resulted in the isolation of two new aromatic tetraols, luisols A and B (Structure 18.13a
and b). Luisol A, formally a reduced hydroquinone, appears to be related to the quinones of the
granaticin class. The structure of luisol B contains the rare epoxynaphtho[2,3c]furan which is only
found in one other natural product, the fungal metabolite anthrinone.

22

Lastly, a

Streptomycete

sp.
was isolated from an estuary near Doheny Beach, California and fermented in a salt-based medium.
The culture broth was found to contain a new pentacyclic polyether, arenic acid (Structure 18.14),
which is related to two known polyether antibiotics, K41-A and oxolonomycin. The structure of
arenaric acid was established by spectroscopic methods involving comprehensive two-dimensional
NMR measurements.

23

Although marine actinomycetes are the most prolific source for bioactive metabolites from
shallow sediments, marine

Bacilli

spp. have also been isolated, and unusual secondary metabolites
have been reported. For example, a

Bacillus

sp. was isolated from marine sediment and cultured
in an enriched seawater medium to yield a new isocoumarin, PM-94128 (Structure 18.15).

24

Comparison of IR and UV data with known substances and one-dimensional

1

H and

13

C NMR
data and a

1

H–

1

H COSY suggested a dihydroisocoumarine derivative with a prenyl group substi-
tuted with a ketide extended leucine. PM-94128 was quite potent with cytoxicity against several
tumor cell lines (IC

50

0.05

µ

M to P388, A-549, HT-29, and MEL-28) and may act by inhibiting
protein synthesis.

B. D

EEP

-S

EA

M

ARINE

S

EDIMENTS

Many interesting metabolites have also been isolated from deep-sea sediments.

2

The energy input
to the sea floor below a depth of 2 km is thought to be less than 10% of the primary productivity
in the euphotic zone. At these depths, food consists of a slow rain of fecal pellets and zooplankton
along with the carcasses of larger organisms from the nekton. Most of these carcasses are consumed
quickly by fish who scatter the remains in the form of feces over large areas to be utilized by
benthic microorganisms. A bacterial isolate from deep-sea mud collected at a depth of 3300 m
off the Aomori coast of the Japan Sea required a seawater medium to grow and produce bioactive
substances. Even though the strain was isolated at 700 atm pressure, it appeared to grow well at
surface pressure and temperature. The strain was identified as

Alteromonas haloplanktis

and was
found to produce a new bioactive siderophore metabolite called bisucaberin (Structure 18.16).

25

This compound had little cytotoxicity but, when added to a mixed macrophage-tumor cell culture,
induced macrophage-mediated cytolysis of tumor cells. Its dimeric structure contains two hydrox-
amates and two amide functionalitites and is similar to other siderophores such as nocardimin
and desferioxamine.
While screening for antitumor effects, Fenical et al.

26
isolated a deep-sea bacterium from a

sediment sample obtained from a 1000-m depth along the California coast. Fermentation of the
slow-growing bacterium in a salt-based medium yielded a series of novel cytotoxic and antiviral
macrolides, the macrolactins A–F. This bacterium was an unidentified Gram-positive organism that
produced six macrolides and two open-chain hydroxy acids when fermented in the presence of salt
at atmospheric pressure. Macrolactin A (Structure 18.17) was the predominate compound produced,
showing moderate antibacterial activity, yet it was quite potent against B16-F10 murine melanoma
in vitro with an IC
50
of 3.5 µg/ml. Of potentially greater significance, macrolactin A inhibited
several viruses, including Herpes simplex (IC
50
= 5.0 µg/ml) and HIV, the human immunodeficiency
virus (IC
50
= 10.0 µg/mL).
Two new caprolactams were isolated by Davidson and Schumacher
27
from an unidentified Gram-
positive bacterium cultured from deep-sea sediments. The caprolactams A and B (Structure 18.18a
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Metabolites of Free-Living, Commensal, and Symbiotic Benthic Marine Microorganisms 575
and b) were obtained as an inseparable mixture and were composed of cyclic-L-lysine linked to
7-methyloctanoic acid and 6-methyloctanoic acid, respectively. Natural products containing a
cyclized lysine are uncommon and have only been reported in several sponges and fungi. In fact,
these structures are quite similar to the bengamides, which are sponge-derived caprolactams with
oxidized acyl side-chains. Thus, one may speculate that the bengamides isolated from sponges may
truly be produced by a symbiotic microorganism. The compounds are mildly cytotoxic toward
human epidermoid carcinoma and colorectal adenocarcinoma cells with MIC values of 10 and
5 µg/mL, respectively, and exhibit antiviral activity toward Herpes simplex type II virus at a

concentration of 100 µg/mL. The same group also isolated a new pluramycin metabolite, γ-
indomycinone (Structure 18.19), from a Streptomyces species isolated from a deep-sea sediment
core sample.
28
γ-Indomycinone is composed of an anthraquinone-γ-pyrone nucleus with a 1-
hydroxy-1-methylpropyl side-chain. The compound exhibits only mild cytotoxicity against a human
colon cancer cell line HCT-116 but shows a differential cytotoxicity against the Chinese hamster
ovary cell lines UV20 (deficient in DNA excision repair) and BR1 (proficient in DNA repair).
These results suggests that γ-indomycinone may act by forming a bulky DNA adduct.
Another Streptomycete species was isolated from deep-sea sediments collected at 1500 m in
the sea surrounding Tokyo, Japan. When this culture was fermented in the presence of seawater,
O
O
OH
N
H
O
OH
OH
NH
2
18.15: PM-94128
O
O
O
O
O
CH
3
O

CH
3
CH
3
OH
H
HH
H
CH
3
OCH
3
CH
3
CH
3
OCH
3
H
H
OCH
3
CH
3
OCH
3
CH
3
OH
COOH

HO
H
18.14: Arenic acid
O
OH
HO
HO
OH
H
O
O
H
H
H
OH
HO
HO
H
H
OO
HO
18.13b: Luisol B
18.13a: Luisol A
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576 Marine Chemical Ecology
growth was enhanced as was the production of two β-glucosidase inhibitors which have the
potential to be developed as antimetastasis or anti-HIV drugs. The two inhibitors were character-
ized as D-glucono-1,5-lactam and D-mannono-1,5-lactam (Structure 18.20). The two lactams had
never been isolated from nature but were found in marine organisms, suggesting that they are of

microbial origin.
29
Simidu et al.
30
isolated 49 bacterial strains from deep-sea core sediments collected at a depth
of 4000 m and examined them for the production of tetrodotoxin. This study indicates that tetro-
dotoxin-producing bacteria are not restricted to certain taxonomic groups. A variety of groups of
bacteria, including Bacillus, Micrococcus, Acinetobacter, Aeromonas, Alteromonas, Moraxella,
Vibrio, and one unidentified bacterium, all produce tetrotoxin. Although the strains are limited in
number, the tetrodotoxin-producing bacteria are quite widespread among various bacterial groups
in marine sediments. It has been postulated that the tetrodotoxins are synthesized solely by bacteria
in sediments and subsequently accumulated by benthic organisms, such as fish and crabs, that
acquire them through the food web.
Another cyclic peptide, halobacillin, was isolated from a marine-derived Bacillus sp. isolated
from a deep-sea sediment core.
31
Halobacillin (Structure 18.21) was only produced in sea-water-
based media and is similar in structure to the surfactins and iturins. Interestingly, the compound
exhibits cytotoxicity against the human colon tumor cell line HCT-116 but lacks the antibacterial
activity associated with surfactin.
H
N
HO
OH
OH
OH
O
18.20: D-Glucono-1,5-lactam
N
N

H
H
N
N
OH
HO
O
O
O
O
18.16: Bisucaberin
HO
HO
O
OH
O
18.17: Macrolactin A
HN
O
H
N
O
R
18.18a: Caprolactin A, R = (CH
2
)
5
CH(CH
3
)

2
18.18b: Caprolactin B, R = (CH
2
)
4
CH(CH
3
)CH
2
CH
3
O
OH O
O
CH3
OH
O
18.19: γ-indomycinone
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Metabolites of Free-Living, Commensal, and Symbiotic Benthic Marine Microorganisms 577
III. COMMENSAL MARINE MICROORGANISMS
Commensal marine bacteria inhabit surfaces, tissues, and internal spaces of other organisms and
plants, but, often, the exact associations are transient and the interactions are not well understood.
Since bacteria occur in seawater at concentrations of one million cells per milliliter, marine plants
and animals are constantly exposed to extremely high concentrations of bacteria. Many of these
bacteria are motile, chemotactic, and/or pathogenic, and readily colonize a variety of surfaces.
Bacteria are the first detectable microorganisms to colonize a surface, which they do by two
processes. The first process is an instantaneous but reversible adsorption of the bacteria onto a
surface from which the bacteria can easily be removed.

32
The second process is an irreversible
adsorption, in which different populations develop with time. These bacterial films often play a
vital role in the growth and development of macroscopic marine plants. A number of seaweeds
have distinctive epibacterial floras that may supply growth factors and contribute to the destruction
of algal autoinhibitory substances. Animal surfaces also appear to selectively enhance or inhibit
microbial colonization. Documentation of these coexistances is increasing, but evidence supporting
true symbiosis rather than a nonobligate association is still lacking. Nonetheless, these associations
are significant in the quest for new bioactive natural products because of the potential cooperative
role in the production of novel metabolites. These surfaces are richer in nutrients than seawater
and most sediments, thus providing a unique niche for the isolation of many diverse bacteria, as
described below.
32
Umezawa and co-workers screened fermentation broths containing various bacterial species,
isolated from the surface of seaweeds, for the production of polysaccharides.
33
One genus, Fla-
vobacterium uglignosum, was found to produce marinactan, which is capable of suppressing
sarcoma-180 tumors in mice. At daily doses of 10 to 50 mg/Kg in mice, marinactan inhibited
75–95% of the growth of these tumors. Marinactan is a neutral heteroglycan consisting of fucose,
mannose, and glucose. In an antibacterial screening program by Sano et al.,
34
a Pseudoalteromonas
sp. was found to produce the novel antibiotic korormicin (Structure 18.22). The organism was
isolated from the tropical green alga Halimeda sp., collected in Palau and required a seawater-
based medium for growth and survival. Korormicin is a combination of an oxidized fatty acid and
an unusual lactonized amino acid. This unusual amino acid is without biosynthetic precedent and
poses an interesting question as to its origin. Interestingly, korormicin was harmless to terrestrial
bacteria and Gram-positive marine bacteria, but was active against 11 marine Gram-negative
bacteria. During the course of an anticancer screening program, a new marine bacterium, Pelagio-

bacter variabilis, was isolated from the blades of the tropical brown alga Pocockiella variegata.
35
This Gram-negative, pleomorphic, halophillic bacterium represents a new genus of marine eubac-
teria. Fermentation extracts of this culture were found to contain the known compound, griseolutic
acid, and three new phenazine antibiotics, the pelagiomicins A–C. Pelagiomicin A (Structure 18.23)
demonstrated strong antibacterial activity to several Gram-negative and Gram-positive bacteria and
inhibited several cancer cell lines in vitro [ID
50
values between 0.04 to 0.2 µg/mL].
Interest in finding new antibiotics against multidrug resistant Mycobacterium tuberculosis and
M. avium-intracellulare led Andersen et al.
36
to isolate one Pseudomonas sp. from the surface of
an unidentified leafy red alga collected in Masset Inlet, British Columbia, and another Pseudomonas
sp. from an unidentified tube worm collected near Moira Island, British Columbia. When cultured
on solid agar in the presence of salt, the two strains produced the novel cyclic depsipeptides,
massetolides A–H, and the known compound viscosin. Massetolide A (Structure 18.24) was two
to four times more potent than viscosin in its in vitro inhibition of M. tuberculosis (MIC = 5–10
µg/mL) and M. avium-intracellulare (MIC = 2.5–5.0 µg/mL), and a single intraperitoneal injection
of 10 mg/Kg of massetolide A was found to be nontoxic to mice. No activity was observed for
either compound against a panel of other human pathogens. A number of unnatural and intriguing
massetolides were produced by feeding with various leucine analogs (L-butyrine, L-norvaline,
L-cyclopropylalanine, etc.) but the supply was not sufficient for testing. The same group also isolated
9064_ch18/fm Page 577 Tuesday, April 24, 2001 5:30 AM
© 2001 by CRC Press LLC
578 Marine Chemical Ecology
a family of novel cyclic decapeptide antibiotics, loloatin A–D (Structure 18.25a–d) from a Bacillus
sp. isolated from a subtidal tube worm collected off Loloata Island, Papua, New Guinea.
37
The

Bacillus sp. was cultured on solid agar containing salt, and the extract from the cells was found to
produce related antibiotics that were active against several strains of antibiotic resistant bacteria.
Loloatins A–C inhibited the growth of methicillin-resistant S. aureus, vancomycin-resistant Entero-
coccus sp., and penicillin-resistant Streptococcus pneumoniae with MIC of 0.5 to 4 µg/mL. Inter-
estingly, only loloatin C exhibited antibacterial activity against the Gram-negative bacterium
Escherichia coli (MIC 1 µg/mL), and loloatin D was four times less active than A–C against Gram-
positive bacteria. These results demonstrate that subtle changes in cyclic decapeptide structure can
have a significant impact on antimicrobial activity. Even though the loloatins share structural
n
-C
7
H
15
H
N
N
H
H
N
O
O
H
N
O
NH
O
NH
O
HN
O

HN
O
NH
O
O
O
O
OH
OH
COOH
OH
18.24: Massetolide A
N
H
H
N
NH
NH
N
H
HN
O
O
ONH
2
O
O
O
O
COOH

O
O
H
n
-C
12
H
25
O
18.21: Halobacillin
O
O
N
H
OOH
O
18.22: Koromicin
N
N
O
OH
O
CH
3
O
O
NH
2
CH
3

CH
3
OH
H
18.23: Pelagiomicin A
9064_ch18/fm Page 578 Tuesday, April 24, 2001 5:30 AM
© 2001 by CRC Press LLC
Metabolites of Free-Living, Commensal, and Symbiotic Benthic Marine Microorganisms 579
features with the tyrocidines, the latter have never been reported to demonstrate Gram-negative
antibacterial activity.
Shigemori et al.
38
isolated a new cyclic alkaloid, alteramide A (Structure 18.26), produced by
an Alteromonas species isolated from the marine sponge Halichondria okadai collected near Nagai,
Kanagawa, Japan. Alteramide A is a macrocyclic lactam containing dienone and dienoyltetramic
acid functionalities that can undergo a [4 + 4] cycloaddition to generate a hexacyclic derivative.
Alteramide A exhibited cytotoxicity against murine leukemia P388 cells, murine lymphoma L1210
cells, and human epidermoid carcinoma KB cells. Related macrocylic lactams, such as ikaruga-
mycin and discodermolide, have been previously isolated from the terrestrial Streptomyces phae-
ochromogenes var. ikaruganesis and the sponge Discodermia dissoluta, respectively, but not from
symbiotic bacteria. The isolation of alteramide may provide insights into the metabolic origin of
discodermolide.
Investigations of microorganisms associated with the Antarctic sponge Isodictya setifera led
to the isolation of a strain of Pseudomonas aeruginosa that exhibited Gram-positive antibacterial
activity.
39
Fractionation of the culture broth identified a new diketopiperazine, cyclo-(L-proline-
L-methionine) (Structure 18.27), five known diketopiperzines, and two known phenazine alka-
loids. Investigations of the sponge revealed that neither metabolite was present. This suggests
H

N
O
O
N
O
NH
O
N
H
O
N
H
O
N
H
O
HN
O
HN
O
N
H
O
N
H
NH
2
OH
HOOC
O

NH
2
R
1
R
2
X
OH
R
2
=
18.25a: Loloatin A R
1
=
X=H
18.25b: Loloatin B R
1
=
N
H
X=H
R
2
=18.25c: Loloatin C R
1
=
N
H
R
2

=
N
H
X=H
18.25d: Loloatin D R
1
=
N
H
R
2
=
X=OH
9064_ch18/fm Page 579 Tuesday, April 24, 2001 5:30 AM
© 2001 by CRC Press LLC
580 Marine Chemical Ecology
that either the bacterium only produces these secondary metabolites under certain environmental
or seasonal conditions, or that the bacterium is present in low abundance in the sponge. Oclarit
et al.
40
isolated a halophytic marine bacterium, Janthinobacterium sp., from an unidentified species
of the Japanese sponge Adocia. Fermentation of this culture produced the compound o-aminophe-
nol (Structure 18.28), which exhibited strong antibacterial activity against Staphylococcus aureus
and Bacillus subtilis. However, a comparison of the antimicrobial activity produced by the
bacterium to the antimicrobial activity associated with extracts of the host sponge revealed that
the two were not related.
In the course of screening for antitumor and immunosuppressive agents, Acebal et al.,
41
from
Pharma Mar, isolated two strains of Agrobacterium sp. from the tunicates Ecteinascidia tubinata

collected in the mangroves of Florida and Polycitonidae sp. collected on the Turkish coast. Ectein-
ascidia turbinata is the source for the very potent antitumor agent ecteinascidin. Fermentation of
these two strains in enriched seawater medium produced lipid-soluble products with potent antitu-
mor activity, sesbanimide A (Structure 18.29) and C. Of interest is that sesbanimides were originally
isolated from the seeds of Sesbania drummondii and Sesbania punicea, leguminous plants. This
isolation from a microbial source confirms their correct metabolic origin and allows for the further
investigation of sesbanimide A, which has immunosuppressive activity. The same group also
investigated the same marine tunicate, Ecteinascida turbinata, collected in Formentera Island,
Spain.
42
Isolation and fermentation of the microorganisms associated with this tunicate revealed
that an Agrobacterium sp. produced a new cytotoxic compound. The compound was obtained from
the bacterial cells by solvent extraction and purified by silica gel chromatogoraphy. Structural
elucidation identified the compound as a new thiazole alkaloid substance called agrochelin and its
acetyl derivative. Agrochelin (Strucure 18.30) is structurally related to yersiniabactin and yersin-
iophore produced by Yersinia enterocolitica and micacocidins A, B, C, isolated from a Pseudomonas
sp. The IC
50
value of agrochelin to P-388 cells was 0.053 µM, which was similar to the value
obtained against mouse lymphoid, human lung colon carcinomas, and human melanoma in vitro
cell lines. The cytotoxic activities of the acetyl derivative were substantially reduced compared
with those of agrochelin.
Molluscs have the potential to be particularly rich sources of microorganisms since they prefer
to feed on the top milliliter of the surface sediment and carry out a high degree of food sorting in
the mantle.
43
It is thought that 97% of the sediment in the mantle is rejected as pseudofeces leaving
N
S
OH

N
S
OH
O
OH
H
H
H
18.30: Agrochelin
H
N
NH
H
H
H
H
H
HO
OH
O
O
O
OH
18.26: Alteramide A
N
NH
SCH
3
H
H

O
O
18.27:
Cyclo
-(L-proline-L-methionine)
OH
NH
2
18.28:
o
-aminophenol
O
O
NH
O
OH
OH
O
O
18.29: Sesbanimide A
9064_ch18/fm Page 580 Tuesday, April 24, 2001 5:30 AM
© 2001 by CRC Press LLC
Metabolites of Free-Living, Commensal, and Symbiotic Benthic Marine Microorganisms 581
an enriched microbial population as a source of food.
43
Because of this sorting process, and because
of microbial digestion, microorganisms are more abundant in the stomach contents of these deposit-
feeders. Kobayashi et al.
44
isolated a Flavobacterium sp. from the marine bivalve, Cristaria plicata,

that was collected at Ishikari Bay, Hokkaido, Japan. The culture was grown statically in enriched
seawater medium and then extracted with organic solvent to yield two new sulfonolipids, flavoc-
ristamide A (Structure 18.31) and its 3,4-dihydro-analog flavocristamide B. Both flavocristamides
exhibited inhibitory activity against a eukaryotic DNA replication enzyme, DNA polymerase α
(IC
50
~ 15–20 µg/mL). Since these compounds are related to ceramide, it appears that the sulfonate
group is important for the activity, since ceramide has been reported to have no effect on DNA
polymerase α. In another study, a marine bacterium, Bacillus cereus, was isolated from the
surface of a toxic snail collected in Izu Penisula, Japan.
45
Fermentation extracts produced from
this culture were discovered to exhibit potent cytotoxicity activity. Two compounds, cereulide
(Structure 18.32a) and homocereulide (Structure 18.32b), were isolated from the lipid extract that
showed potent activity against P388 and Colon 26 tumor cell lines with an IC
50
of 1 and 35 pg/mL,
respectively. The B. cereus strain was never proven to be the source of the poisoning produced by
the snail.
Actinomycetes have also been isolated from the relatively nutrient-rich sufaces of invertebrates
and seaweeds.
6
Fenical et al.
46
isolated an unidentified Actinomycete from the surface inoculum of
the Carribean brown alga Lobophora variegata that produced two new macrolides, lobophorins A
and B (e.g., Structure 18.33). Despite their structural relationship to kijanimicin and to several
related antibiotic macrolide glycosides such as tetrocarcins and chlorothricin, lobophorins A and
B did not exhibit significant antibiotic properties. However, they did show potent antiinflammatory
activities in the phorbol-myristate-acetate-induced mouse ear edema model. At the normal testing

dose, lobophorin A and B reduced edema by 86% and 84%, respectively, and lobophorin B
demonstrated in vivo activity when administered intraperitonly in mice.
Fenical’s group
47
has also investigated actinomycetes living on the surface of marine inverte-
brates. They isolated a streptomycete from the surface of a gorgonian coral (Pacifigorgia sp.)
collected from the Gulf of California, Mexico. When fermented in marine media, the isolate
produced several metabolites, including the 20-hydroxy derivative of oligomycin A, the 5-deoxy
derivative of enterocin, and the octalactins A and B. The octalactins belong to a new structure class,
which are C
19
ketones possessing rare eight-membered ring lactone functionalities. Octalactin A
(18.34) demonstrates potent in vitro cyctoxicity against B16-F10 murine melanoma and HCT-116
human carcinoma cell lines. The surface of a tropical jellyfish, Cassiopeia xamachana, yielded a
Streptomyces sp. that produced two new bicyclic peptides, salinamides A (Structure 18.35) and B,
which have novel depsipeptide backbones.
48
The salinamides A and B exhibit activity against all
Gram-positive microorganisms tested. Interestingly, salinimide A and B also demonstrate potent
anti-inflamatory effects (84% inhibition at 50 µg/ear) in the inhibition of phorbol-ester-induced
edema in the mouse ear model. Recently three additional minor peptides were isolated from this
culture. Salinamides C and E are monocyclic depsipeptides that are likely methylated byproducts
of salinamide A biosynthetic intermediates, and salinamide D contains a D-valine residue in place
of the D-isoleucine moiety in salinamide A.
49
Research at Pharma Mar resulted in the isolation of a new species of Micromonospora from
an unidentified marine soft coral collected off the coast of Mozambique.
50
This culture produced
a novel depsipeptide designated PM-93135 when fermented in a nutrient-rich medium. PM-93135

exhibited antibacterial activity against S. aureus, B. subtilis, and Microcoous luteus and inhibited
RNA synthesis in P388 cells with an IC
50
of 0.008 µg/mL. In addition, this compound demonstrated
significant antitumor activity against P388, human lung carcinoma A-549, human colon carcinoma
HT-29, and human melanoma MEL-28 with IC
50
of 0.0002, 0.002, 0.01, and 0.0025 µg/mL,
respectively. Another study involved the isolation of a Streptomyces sp. from the surface of a mollusc
collected in Kanagawa Prefecture, Japan.
51
This culture, when fermented, produced a novel cyto-
toxic agent that induced apoptotic cell death. The compound, aburatubolactam C (Structure 18.36),
9064_ch18/fm Page 581 Tuesday, April 24, 2001 5:30 AM
© 2001 by CRC Press LLC
582 Marine Chemical Ecology
O
O
O
O
O
NH
NH
2
H
H
O
O
O
O

OH
OH
CH
3
O
O
HO
HO
O
H
H
H
O
O
OH
18.33: Lobophorin A
O
H
N
O
O
O
HN
O
O
O
NH
O
O
O

H
N
O
O
O
HN
O
O
O
NH
O
O
R
18.32a: Cerulide, R=H
18.32b: Homocerulide, R=CH
3
HO
3
S
HN
O
OH
OH
10
8
18.31:
Flavocristamide A
9064_ch18/fm Page 582 Tuesday, April 24, 2001 5:30 AM
© 2001 by CRC Press LLC
Metabolites of Free-Living, Commensal, and Symbiotic Benthic Marine Microorganisms 583

has a novel lactam structure consisting of a 20-membered macrocycle coupled with a unique acyl
tetramine and bicyclo[3.3.0]octane. It is related to several terrestrial Actinomycete products as well
as alteramide A, which was produced by an Alteromonas sp. isolated from a sponge.
Aburatubolactam C was cytotoxic for several proliferating tumor cells of human and murine origins
with IC
50
of 0.3 to 5.8 µg/ml. When Jurkat T cells were treated with 3 µg/mL of the compound,
the apototic DNA fragmentation was detectable in 3 hours, indicating that the effect of aburatubo-
lactam C was attributable to induced apoptosis.
IV. SYMBIOTIC MARINE MICROORGANISMS
Even though symbiosis is a widespread phenomenon and an important agent of evolution, little is
known about symbiotic relationships between prokaryotic and eukaryotic taxa.
52
The endobiotic
environment is comprised of cells of microorganisms as well as the tissues of plants and animals
that serve as hosts to a wide spectrum of microbial forms in a variety of relationships. These
relationships include mutualism (organisms of different species live together for the benefit of both),
parasitism (only the parasite derives nourishment from the host, but does not necessarily cause
O
O
H
N
O
N
OH
O
18.36: Aburatubolactam C
O
O
NH

O
H
N
O
NCH
3
O
O
N
H
O
O
O
O
N
H
HN
O
HO
H
N
H
O
HO
O
H
H
18.35: Salinamide A
O
HO

O
O
O
OH
18.34: Octalactin A
9064_ch18/fm Page 583 Tuesday, April 24, 2001 5:30 AM
© 2001 by CRC Press LLC
584 Marine Chemical Ecology
disease), and pathogenesis (the pathogen can cause disease in the host). Mutualistic symbiosis and
disease are very important factors that affect the ecology of both microorganisms and macroorgan-
isms in the sea. Many of these associations began as incidental interactions that later developed
into a relationship of obligate mutualistic symbioses. This range of interdependence includes a
wide spectrum of bacterial/host adaptations, including the biosynthesis and maintenance of unique
secondary metabolites. Among the first conclusive reports of marine symbiosis are relationships
between chemoautotrophic bacteria and marine invertebrates in deep-sea hydrothermal vents. The
hydrothernal vents emit sulfide, which, in turn, provides the necessary energy and reducing power
for chemoautotrophic bacteria. Many of these bacteria live in symbiotic asssociations with macro-
invertebrates living in close proximity to these vents, sometimes directly providing their sole source
of nutrition. Furthermore, some of the invertebrates living there either contain a greatly reduced
digestive tract or have no digestive tract at all, forcing them to rely completely upon these endo-
symbionts for their survival.
52
Bacterial symbionts that are chemoheterotrophic have been described and isolated in pure
culture. Fenical et al.
53
studied the resistance of the estuarine shrimp Palaemon macrodactylus to
pathogens and observed that their eggs harbored bacterial epibionts. Upon removal of the bacterial
epibionts, rapid infestation of the pathogenic fungi Lagenidium callinectes occurred. Further inves-
tigation revealed that a penicillin-sensitive Alteromonas species could be consistently isolated from
the healthy embryos. This bacterial strain was fermented and found to produce a potent antifungal

agent, 2,3-indolinedione, also known as istatin. Bacteria-free embryos could become disease-free
if they were either reinoculated with the bacteria or treated with istatin. Interestingly, istatin had
been known for many years as a synthetic intermediate in the production of indigo dyes but not
as an antifungal agent. Gil-Turnes et al.
54
discovered a similar relationship after investigating the
eggs of the American lobster Homarus americanus. The eggs were found to be colonized by an
unidentified unicellular bacterium that produced the phenolic compound tyrosol (2-p-hydroxyphe-
nol ethanol). The bacterium produces this phenol in quantities which are sufficient to control
pathogenic microorganisms.
Some of the best examples of chemistry derived from marine microbial symbionts are those
microorganisms responsible for producing many of the marine toxins that pose human health
hazards. One of these, surugatoxin (Structure 18.37), which specifically blocks nicotinic receptors
and is a causative agent of shellfish poisoning in Japan, was initially isolated from the gut of the
Japanese Ivory Shell snail Babylonia japonica.
55
Because of the seasonal outbreaks of this toxin
and the ability of the snails to become toxin-free after being introduced to a new environment, it
was proposed that a microorganism may be the source of this poison. Subsequently, neosurugatoxin
and prosurugatoxin, precursors of surugatoxin, were isolated from a Gram-positive Coryneform sp.
obtained from the mid-gut of B. japonica.
N
H
N
H
N
NH
H
N
Br

HO
HO
OH
OH
OH
O
O
HO
O
O
O
OH
CH
3
18.37: Surugatoxin
9064_ch18/fm Page 584 Tuesday, April 24, 2001 5:30 AM
© 2001 by CRC Press LLC
Metabolites of Free-Living, Commensal, and Symbiotic Benthic Marine Microorganisms 585
Marine sessile invertebrates are considered a particularly rich source of novel metabolites and
symbiotic microorganisms.
2
Sponges, for example, may have up to 40% of their cellular volume
occupied by associated bacteria. The surfaces, tissues, and internal spaces of such invertebrates
provide a variety of marine microhabitats and harbor tremendous potential for diverse microorgan-
isms. Bacteria associated with marine invertebrates experience a wide range of environmental
parameters including variable pH, nutrient availability, and surface texture. Collectively, these
factors may select for novel biosynthetic pathways for survival. Jackson and Buss
56
have suggested
that many cryptic marine invertebrates have evolved species–species allelochemical effects against

competitors. Such allelochemicals are thought to play an important role in the ecology of many
algae and benthic invertebrates. Studies of sponges and holothurians have suggested that fish
predation and grazing play an important role in selecting for toxicity in coral reef invertebrates.
Crypticity may have evolved as a means of protection for nontoxic shell-less invertebrates, while
toxic species may live both unexposed and exposed to fish predation and grazing.
56
These chemical
defenses, which may be attributed to the associated symbiotic microorganisms can be quite impor-
tant mediators of predation, competition, or epizoic colonization.
2
However, identification of the
symbiotic microorganism(s) potentially responsible for the production of a specific defensive
metabolite can be difficult. Often, only circumstantial evidence obtained from the marine inverte-
brate is presented to indicate that the metabolite(s) originated from a symbiotic microorganism.
Such evidence usually consists of extremely low or variable metabolite yields, isolation of identical
metabolites from different sources, or similarity in compound structure to metabolites already
described from another microorganism.
57
In retrospect, failure to isolate the desired metabolites
from microbial symbionts is not surprising. It is well known from studies of terrestrial microor-
ganisms and now marine microorganisms, that, to be productive, the symbionts may require host-
specific growth conditions, and that often these physiological parameters remain unknown.
57
Sponges have provided more natural products with unprecedented molecular structure and
bioactivities than any other phylum of marine invertebrates. In many poriferans, complex microbial
communities have been described that contribute to the lives of their hosts.
58,59
In addition to
providing a primary food source, microorganisms can also process waste products, transfer nutri-
ents, produce reef-like structures, or produce secondary metabolites.

58,59
However, despite the
numerous associations documented between sponges and microbial symbionts, most microorgan-
isms remain uncultured or unculturable.
57
This remains a major challenge, and new methods of
isolation that include a consortia of microorganisms in combination with growth conditions that
mimic the in situ environment must be developed. In the 1970s through 1980s, descriptions of
symbioses relied on microscopy to demonstrate the presence of specific symbionts or chemical
measurements of nutrient transfer. Recently, molecular phylogenetic surveys have proven the
existence of these uncultivated marine microorganisms. One example is the discovery and prelim-
inary characterization of a marine archaeon that inhabits the tissues of a temperate water sponge.
60
The microorganism, Cenarchaeum symbiosum (Phylum: Crenarchaeota), inhabits a single species
of sponge, Axinella, and grows well at temperatures of 10°C, which is over 60°C below the growth
temperature optimum of any cultivated archeon species. In situ hybridization studies concluded
which microorganism in the sponge was archael and allowed localization of the symbiont. The
high abundance of a single, crenarchaeal phylotype in every specimen of Axinella sponge examined
and the presence of active cell division in laboratory-maintained sponges over long time periods
strongly suggest that this partnership is a true symbiosis.
The first definitive example of a sponge natural product being derived from its associated
microorganism was demonstrated by Oclarit and co-workers.
61
They isolated a Vibrio species from
the homogenate of the marine sponge Hyatella sp. collected along the coast of Oshima Island,
Miyazaki, Japan. When this bacterium was cultured on marine agar, it was found to produce the
compound andrimid (Structure 18.38a). This same bioactive compound was also found in the sponge
extract, suggesting that the active component may be synthesized by the associated microorganism.
Adrimid had been previously isolated from the culture of an Enterobacter sp. that is an intracellular
9064_ch18/fm Page 585 Tuesday, April 24, 2001 5:30 AM

© 2001 by CRC Press LLC
586 Marine Chemical Ecology
symbiont of the Brown Planthopper Nilaparvata lugens and was found to exhibit potent activity
against Xanthomonas campestris pv. oryzae. Needham et al.
62
also isolated andrimid and the
moiramides A–C (e.g., Structure 18.38b), but from a marine Pseudomonas fluorescens isolated
from an unidentified Alaskan tunicate. Andrimid and moiramide B both potently inhibited the
growth of methicillin resistant S. aureus. Due to the diversity of the microorganisms producing this
toxin, one can speculate that the production of this compound can be encoded by genes transferable
on a plasmid.
Numerous examples exist in which structurally related or identical compounds have been
reported from taxonomically distinct marine invertebrates or from animals collected in certain
geographic locations.
63
The microbial-type structures of compounds isolated from marine sponges
and ascidians lends support to the hypothesis that many marine invertebrate natural products may
be of microbial origin. One of the most striking examples is the isolation of a new endiyne antitumor
antibiotic called namenamicin, (Structure 18.39) isolated from the didemnid ascidian Polysyncraton
lithostrotum.
64
Members of this class of compounds were first isolated from terrestrial actinomycetes
in the genera Micromonospora and Actinomadura, which produce calicheamicin γ (Structure 18.40)
and esperimicin, respectively.
65
Additional examples include the isoquinoline alkaloids such as
ecteinascidin (Structure 18.41) and renieramycin (Structure 18.42). These compounds have been
isolated from the ascidian Ecteinascidia tubinata and the sponge Reiniera sp., with the terrestrial
counterpart saframycin B (Structure 18.43), isolated from Streptomyces lavendulae.
66

Faulkner et al.
67
have proposed that secondary metabolites are biosynthesized within the cells
they are localized and have demonstrated that these metabolites are cellularly located within
symbiotic microorganisms. Due to the failure of culturing symbiotic microorganisms, cellular
localization studies have become a model of evaluating invertebrate–microbial symbioses and the
production of microbially derived natural products. The chemically productive sponge Theonella
swinhoei is well known for harboring a diverse series of biologically active secondary metabolites,
many of which resemble microbial products. Bewley and Faulkner
68
analyzed the origin of these
natural products by conducting separate chemical isolations from the various cell populations
isolated from the intact sponge. The cells were dissociated with a “juicer” and purified by differential
centrifugation. Four distinct populations were recovered: sponge cells, heterotrophic unicellular
bacteria, filamentous bacteria, and from the ectosome, unicellular cyanobacteria. These were sep-
arately extracted and chemically analyzed. The macrolide swinholide A (Structure 18.44) was found
to occur only in the heterotrophic unicellular bacteria, while the bicyclic peptide theopalauamide
(Structure 18.45) was localized in the filamentous bacteria. These results were unexpected since
both these metabolites have structural precedent as cyanobacterial products. Another unexpected
result was that no major secondary metabolites were found in the extracts of either the sponge or
the cyanobacteria. This work has raised as many questions as it has answered. Contrary to earlier
speculation, the identification of the microorganisms that produce swinholide A and theopalauamide
remains unknown. One could postulate that the filamentous bacteria have evolved from a cyano-
bacterial origin or that the compounds are synthesized from the cyanobacteria and have diffused
to other regions of the sponge for various self-protective mechanisms. This work clearly charac-
terizes the complexity of the ecology of marine invertebrates and their associated microorganisms
and exemplifies how little we understand about the chemical ecology of marine symbionts.
Another example of a natural product produced via a symbiont results from the work of Haygood
et al.
69

Using the bryozoan Bugula neritina as a model system, their laboratory is investigating
whether the biosynthetic source of the potent bryostatins (Structure 18.46) is a marine microorgan-
ism. The bryostatins are an important family of cytotoxic macrolides based on the bryopyran ring
system, and bryostatin 1 is currently in Phase II clinical trials for the treatment of leukemias,
lymphomas, melanoma, and solid tumors. An important advantage of this system is that the
microbial community of B. neritina consists of a single bacterial symbiont and few other bacteria,
in contrast to the complex microbial populations of most sponges. This symbiont is a rod-shaped
Gram-negative bacterium localized in the larvae of B. neritina. Bryostatin is also found in the larvae
9064_ch18/fm Page 586 Tuesday, April 24, 2001 5:30 AM
© 2001 by CRC Press LLC
Metabolites of Free-Living, Commensal, and Symbiotic Benthic Marine Microorganisms 587
where it may be used for chemical defense. Symbiont ribosomal sequences have been obtained,
and in situ hybridizations have demonstrated that there are approximately 2500 symbionts per larva.
In addition, analyses of sequence from larvae isolated from seven different west coast and one
Atlantic coast population of B. neritina, shows that the same symbiont is present in all cases.
Phylogenetic analysis revealed that the symbiont is a new genus of γ-proteobacterium and has been
named “Candidatus endobugula sertula.” Unfortunately, the symbiont has not been viable in culture,
which may indicate it is a true symbiont. However, bryostatin is a complex polyketide. The
biosynthesis of complex polyketides is well understood and the molecular biology and cloning of
these pathways is a very active area of research.
70
It may be quite feasible to clone the biosynthetic
genes of bryostatin and solve the problem of supply without cultivating the microorganism.
N
OAc
O
O
S
OMe
HO

NMe
OH
O
O
NH
MeO
HO
18.41: Ecteinascidin
H
N
H
N
H
OO
O
NH
O
R
O
18.38a: Andrimid, R = H
18.38b: Moiramid C, R = β-OH
O
O
S
HO
CH
3
O
O
NH

HO
S
OH
O
O
H
N
O
OH
O
S
CH
3
CH
3
CH
3
CH
3
S
CH
3
CH
3
S
CH
3
O
O
CH

3
18.39: Namenamicin
OCH
3
O
O
OH
S
O
O
OH
CH
3
OCH
3
O
OCH
3
I
NH
O
OH
O
O
H
HO
O
NH-CH
2
CH

3
H
3
C
CH
3
O
CH
3
NH-CO
2
CH
3
O
HO
CH
3
SSS
CH
3
18.40: Calicheamicin γ
9064_ch18/fm Page 587 Tuesday, April 24, 2001 5:30 AM
© 2001 by CRC Press LLC
588 Marine Chemical Ecology
O
CO
2
Me
O
O

H
OH
OH
O
CO
2
Me
HO
OH
O
O
OH
18.46: Bryostatin
N
O
O
MeO
H
O
OMe
O
OH
O
O
18.42: Renieramycin
NMe
N
O
MeO
H

O
OMe
O
NH
O
O
O
18.43: Saframycin B
NMe
O
O
OH
OH
O
O
OH
OH
O
O
O
OCH
3
HO
HO
O
HO
OCH
3
OH
OCH

3
OCH
3
18.44: Swinholide
H
N
N
H
H
N
NH
O
NH
O
HN
O
N
H
O
HN
O
NH
O
HN
O
HN
O
NH
O
H

2
NOC
O
O
O
N
N
OH
HO
OH
OH
Br
HO
OH
HO
H
2
NOC
OH
HOOC
OH
HO
18.45: Theopalauamide
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© 2001 by CRC Press LLC
Metabolites of Free-Living, Commensal, and Symbiotic Benthic Marine Microorganisms 589
V. SUMMARY
This chapter has presented examples that illustrate the diversity of microorganisms living in the
sea and the plethora of natural products that has been derived from them. The structural and
biosynthetic diversity of these microbial compounds coupled with their pharmacological activities

is truly striking, and, clearly, the potential for discovering new natural products remains quite
promising. However, most of the microorganisms and chemical entities that have been discovered
are the result of traditional protocols. The majority of the eubacteria described have been isolated
on conventional marine agar and cultivated in simple marine fermentation media, either in shake
flasks or on solid agar surfaces. These physiological parameters are quite limited and do not mimic
the natural marine environment. The difficulties encountered in the isolation and cultivation of
microbial symbionts reveal the profound lack of understanding of these complex associations.
Research efforts must focus on developing new isolation and fermentation methods that include a
consortium of microorganisms in combination with growth conditions that are found in the original
marine environment. Studies must also be initiated that focus on the effects of host metabolites
upon the distribution of marine bacteria and the production of natural products. Clearly, exploration
of the marine environment is just beginning, and the potential for discovering new microorganisms
and novel bioactive compounds remains an exciting field of research.
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