Induction of settlement and metamorphosis in Crassostrea virginica by melanin-synthesizing bacteria

The present invention relates to the discovery of a new bacterium, alteromonas calwellii, which has been found to attract oyster larvae by the production of a compound involved in melanin synthesis. More specifically, the present invention contemplates a method for inducing the settlement and metamorphosis of Crassostrea virginica larvae by induction with certain metabolic substances produced by the present bacterium and its mutagenically altered variants. Furthermore, the present invention is directed to other and derivative metabolic products which can be employed for their desired utility and application.

BACKGROUND OF THE INVENTION 
The present invention relates to the discovery of a new bacterium which has 
been found to attract oyster larvae by the production of a compound 
involved in melanin synthesis. More specifically, the present invention 
contemplates a method for inducing the settlement and metamorphosis of 
Crassostrea virginica larvae by induction with certain metabolic 
substances produced by the present bacterium and its mutagenically altered 
variants. Furthermore, the present invention is directed to other and 
derivative metabolic products which can be employed for their desired 
utility and application. 
The formation of pioneer microbial communities on submerged surfaces 
appears to be beneficial to subsequent attachment and development of many 
invertebrate larvae. A number of investigations have established a general 
pattern of periphytic succession for colonization of clean surfaces 
immersed in seawater. In the initial phase after possible coating by 
organic matter, bacteria attach to such a surface and begin to grow, 
forming microcolonies within several hours. Subsequently, diatoms, fungi, 
protozoans, micro-algae and other microorganisms attach to the surface, 
forming what has been termed the primary slime layer. This primary 
microbial colonization appears to be a prerequisite for the final stage of 
succession in which microorganisms, viz., invertebrates, attach and grow 
on the surface. Although most surfaces are eventually colonized, the 
rapidity and extent of the process depends on the nature of the surface 
material, the prevailing environmental conditions and the composition of 
the periphytic populations. 
Two invertebrate species for which some information has been ascertained 
concerning the effect of the periphytic organisms on their induced 
metamorphosis are the sea urchin, Lytechinus pictus, and the hydroid, 
Hydractinina echinata. It has been determined that for Lytechinus, the 
responsible factor is a low molecular weight bacterial by-product, 
probably proteinaceous having a molecular weight less than 5000 daltons. 
It has also been found that planulae larvae of Hydractinia metamorphose in 
response to a product emitted by certain marine, gram-negative bacteria at 
the end of their exponential growth phase. If these bacterial cultures are 
subjected to osmotic shock, the activity shows up in the supernatant, 
suggesting that the critical product is a soluble factor rather than a 
bound one. When Hydractinia are kept in sterile conditions, they do not 
metamorphose. 
In a series of experiments designed to determine the physiological 
mechanism by which the stimulus activates metamorphosis, it has been 
demonstrated that the inducer may operate by stimulating the Na.sup.+ 
/K-ATPase of larval cell membranes. Such findings are the first real steps 
toward understanding how larvae can mount a broad spectrum morphogenetic 
response to specific environmental stimulation. Moreover, recent reports 
have shown that Vibrio sp. excretes a product that induces metamorphosis 
of the cnidarian, Cassiopea andromeda. Other investigations demonstrate 
that larvae of the marine annelid, Janua brasiliensis, settle on certain 
microbial films and that certain specific bacteria may induce 
metamorphosis. These observations suggest that the processes are mediated 
by larval lectins binding to extracellular polysaccharides or 
glycoproteins, produced by the bacteria. 
In both the natural environment and in oyster mariculture operations, the 
setting process, whereby planktonic oyster larvae alight on an oyster 
shell or plastic sheet and undergo metamorphosis to form attached oyster 
spat, is crucial to successful oyster development. It is also known that 
the larvae of Ostrea edulis, the European oyster, prefer setting on 
surfaces covered with a film of bacteria and diatoms. Natural periphytic 
microbial populations are, therefore, significant in successful oyster 
setting. The same situation is likely to be true of oyster mariculture, 
since a rich source of bacterial flora has been associated with oyster 
larvae and larval food sources in hatcheries. In some cases, bacteria have 
also been implicated in the death of oyster larvae. Since the presence of 
microorganisms significantly affects oyster development, improved 
knowledge of the biology of these microorganisms and particularly an 
understanding of their beneficial and/or deleterious effects on developing 
oysters, will further improve oyster setting and development in both 
natural and artificial settings. 
Oyster larvae display three characteristic patterns toward organic 
compounds and microorganisms, i.e., positive, inactive and negative 
chemotaxis. In one particular study, a marine pseudomonad was attractive 
to larvae while a marine yeast elicited no response. It has also been 
suggested that an alga, Isochrysis, may produce extracellular oyster 
attractant. Conversely, it is known that oyster larvae do not set 
preferentially on surfaces to which a marine isolate, Hyphomonas 
neptunium, is affixed. It is believed that H. neptunium does not 
antagonize settlement, but rather that it competitively establishes itself 
on surfaces and excludes bacterial species which would be beneficial to 
oyster settlement. 
The question, however, of which of the periphytic microorganisms and which 
of their products specifically attract or promote the setting and 
subsequent development of oyster larvae has not been answered heretofore. 
Free swimming larvae, shortly after spawning, seek a suitable place to 
settle and attach themselves. A number of environmental conditions are 
involved in settlement, salinity and nutritional availability are probably 
the most important. But once larvae are satisfied with these initial 
conditions, they appear to respond to a biochemical cue to settle and 
attach themselves. That biochemical cue is released by a pigmented 
bacterium which adheres strongly to surfaces such as oyster shells and 
which is the subject of this invention. 
SUMMARY OF THE INVENTION 
It is, therefore, one object of the present invention to provide a marine 
bacterium which is capable of inducing the settlement and metamorphosis of 
Crassostrea virginica larvae. 
Another object of this invention is to provide a method for inducing the 
settlement and metamorphosis of Crassostrea virginica larvae. 
A further object of the present invention is to isolate and purify the 
metabolic products of the present bacterium and those of its mutagenically 
altered variants. 
Still another object of this invention is to employ the isolated metabolic 
products of the present bacteria for their desired utility. 
These and other objects are achieved by the discovery of a 
melanin-synthesizing marine bacterium, designated LST, which has been 
mutagenically altered in accordance with this invention to provide two 
particular variants thereof, designated DIF and HYP. These bacteria have 
been taxonomically identified as a new species of the genus Alteromonas, a 
genus established to include a diverse array of polarly flagellated, 
aerobic marine bacteria. These bacteria have been named and characterized 
as Alteromonas colwellii. Each bacterium has been deposited with the 
American Type Culture Collection (ATCC) and have been accorded the 
accession numbers 39565, 33887 and 33888, respectively. LST and its 
variants, DIF and HYP, and/or any of their variants, can be employed in a 
process for inducing the settlement and metamorphosis of Crassostrea 
virginica larvae. This process may be employed in a natural or artificial 
environment, e.g., a mariculture operation, to induce the setting process 
of oyster larvae during which process the larvae alight on cultch or other 
suitable surface materials, and undergo metamorphosis to form attached 
oyster spat. The present method can be effected by exposing the 
Crassostrea virginica larvae to the LST, DIF, HYP and/or any of their 
variant bacteria or the melanin and melanin precursor metabolic products 
thereof in an aqueous environment. 
Moreover, certain metabolic products of the present bacteria can be 
isolated and employed for their desired utility. The DIF and HYP variants 
exhibit particularly heightened levels of production of these metabolic 
substances.

DETAILED DESCRIPTION OF THE INVENTION 
In accordance with the present invention, a new melanin-synthesizing marine 
bacterium, designated LST, has been isolated in continuous and close 
association with oysters and can be employed in a process for inducing the 
settlement and metamorphosis of Crassostrea virginica larvae. Moreover, 
several metabolic products have been recovered from the present bacterium 
which are highly desirable for their industrial, experimental or medical 
utility. 
Thus, among the many advantages of the present invention, it has been 
surprisingly discovered that the novel marine bacterium of this invention 
and, particularly, the mutagenically altered variants thereof, designated 
DIF and HYP, which have also been accorded the ATCC accession numbers 
39565, 33887 and 33888, respectively, are capable of specifically inducing 
the setting and metamorphosis of Crassostrea virginica larvae by the 
production of certain metabolic products associated with melanin 
synthesis. These metabolic products, melanin, pheomelanin and 
dihydroxyphenylalanine have been found to have significant commercial, 
experimental and medical utility. 
In more detail, LST and its related variants are Vibrio-like, aerobic, 
highly motile, gram-negative rods, with a guanosine to cytosine ratio of 
45.6% and have been found to attract oyster larvae by production of a 
compound involved in melanin synthesis. The compound is most abundantly 
synthesized during the late stationary-decline phase of bacterial growth 
when the adenylate energy charge is 0.72 and the organism is undergoing 
morphological transition to the elongated helical form. The bacteria grow 
optimally in 35 ppt salt, and within a range of 15-75 ppt at 25.degree. C. 
and do not produce spores. The bacteria readily attach to a variety of 
surfaces including, preferentially, glass and oyster shells, and also to 
plastic, aluminum, and the like. The biochemical characteristics of LST 
are set forth in Table 1 below. These bacteria have been taxonomically 
identified as a new species, colwellii, of the genus Alteromonas. 
LST, DIF and HYP, though heterotrophic, have relatively simple nutritional 
requirements as set forth below in Table 2. Although serine and methionine 
alone do not support the growth of LST or its related variants, aspartic 
and glutamic acids, in combination with serine, methionine or each other, 
do sustain the organism. Therefore, either aspartic or glutamic acid could 
serve as a carbon and energy source. In practice, however, it is preferred 
that a growth medium of aspartic acid and a solution of inorganic salts be 
supplemented with glutamic acid to remedy the growth-limiting effects 
which may be observed after numerous subculturings. 
LST and its mutagenically altered variants, particularly DIF and HYP, are 
marine bacteria that use amino acids but do not use carbohydrates, fail to 
ferment sucrose, mannose or arabinose (see Table 1), and fail to grow in a 
medium containing 1% glucose and salts solution. 
In the stationary phase of growth, these bacteria routinely produce a 
reddish-brown pigment, which has been identified as a melanin and which 
bio-synthetic pathway has been discovered to mediate the interaction 
between the bacteria and the oyster larvae. Noteably, oyster larvae must 
settle prior to metamorphosis and do so in response to a chemical cue, 
i.e., a positive chemotaxis. In this regard, it has been determined that 
the melanin macromolecule is actually a heteropolymer of a number of 
different monomeric precursors. Of particular importance is 
dihydroxyphenylalanine (DOPA), a melanin precursor which increases oyster 
spat attachment and thus supports the settling of larvae. DOPA is also 
known to have significant physiological applications, primarily acting as 
a neurotransmitter in humans and animals and is often used in the 
treatment of Parkinson's disease or other related nervous disorders. 
Thus, in one aspect of the present invention, two mutagenically altered 
variants of the parent LST bacterium, designated DIF and HYP, have been 
isolated through conventional techniques such as, for example, mutagenesis 
with ethyl methane sulfonate (EMS) or ICR 191 (Institute of Cancer 
Research intercalating agent). Each of these variants exhibit unique 
characteristics relative to the production of the metabolic pigment 
products, e.g., melanin, pheomelanin, DOPA, tyrosine, tyrosinase and 
related enzymes. Each of the variants can therefore be selectively 
employed in a process for the settlement and metamorphosis of oyster 
larvae. By the term variant or mutant is meant the genetic derivative of 
the parent bacterium which is obtained by single or multiple base 
substitutions, deletions, insertions or inversions whether spontaneously 
or artificially induced. 
Moreover, the LST, DIF and HYP strains each produce an additional 
exopolymer as a product of their metabolism. This acid polysaccharide has 
been found to have excellent utility as a marine cement, a water-proofing 
material and an emulsifying agent which can be employed, for example, in 
the clean-up and removal of oil and organic spills. 
In one particular embodiment of this invention, each of the present 
bacteria or their variants can be employed in a process for inducing the 
settlement and metamorphosis of Crassostrea virginica larvae. Accordingly, 
the bacteria are cultured in a growth medium and provided with a suitable 
surface material to which they can affix due to the production of the acid 
polysaccharide exopolymer. Oyster larvae are simultaneously or thereafter 
exposed to the bacteria or, alternatively, to their melanin or melanin 
precursor metabolic products including melanin, pheomelanin, DOPA, or 
mixtures thereof for a time and under conditions to effect larvae setting. 
Once settlement occurs, metamorphosis, i.e., maturation of the oyster 
larvae, naturally progresses in response to the micro-colonies of bacteria 
which develop on the provided surface material. 
By way of explanation, although not wishing to be bound, it is believed 
that when sufficient numbers of bacteria are achieved, during the decline 
phase of growth., the bacterial colonies produce a high concentration of 
pigment, i.e., melanin and the precursor DOPA, which attracts oyster 
larvae. The larvae appears to be able to "ingest" (feed upon) these 
elongated cells (.gtoreq.5 .mu.m) of bacteria which are observed to occur 
during that stage of growth. Moreover, an oyster product appears to induce 
LST reproduction, similar to lectins produced by Halachondrea panicea, 
which stimulate the bacterium, Pseudemonas insolita. The association 
between the present bacteria and oyster larvae may, therefore, involve a 
hormone-like stimulatory effect on, or function involved in, larval 
development and metamorphosis. 
The preferred growth medium for the bacteria of this invention is brain 
heart infusion with about 3% NaCl, although other conventional growth 
media which meet the nutritional requirements set forth above will 
suffice. (Alternative media are set forth in the Examples under Organism 
and Culture Conditions.) It is also preferable that the oyster larvae be 
fed Isochrysis galbana and Monochrysis lutheri at a rate of about 
2.times.10.sup.5 cells/ml of culture per day. The ideal surface upon which 
the bacteria can be grown is cultch, although glass is just as effective 
and more commercially expedient. Materials such as plastics or aluminum 
are also satisfactory. 
Since the LST strain is mutagenically altered in accordance with this 
invention using ICR 191 to obtain the DIF and HYP variants, each variant 
has its own unique characteristics. Specifically, the DIF bacterium, ATCC 
number 33887, produces a low molecular weight pigment (.ltoreq.30,000 
daltons) that readily diffuses into the water column, i.e., any body of 
water. It is preferable, therefore, that this bacterium is employed in a 
process to induce high proportions of oyster larvae to uniformly set on 
varying surfaces to promote enhanced, but undirected, set. Moreover, the 
DIF strain specifically produces increased amounts of pheomelanin. On the 
other hand, the HYP bacterium, ATCC number, 33888, produces amplified 
amounts of melanin and melanin precursors of high molecular weight 
(.gtoreq.100,000 daltons) that do not readily diffuse into the water 
column. It is preferable that the HYP variant is employed in a method to 
induce setting on specific surfaces for high level production of oyster 
development, and for the recovery of higher amounts of the melanin and 
melanin precursor metabolic products such as, for example, melanin, DOPA, 
tyrosine, tyrosinase and related enzymes. 
Notably, each of the bacterial strains produce high amounts of the 
exopolymer, an acid polysaccharide, which can be employed, for example, as 
a marine cement, a water-proofing substance and/or as an emulsifying 
agent. 
In another embodiment of the present invention, a method has been provided 
for the recovery and purification of the pigments metabolically produced 
by the present bacteria. The pigment is a melanin macromolecule, a 
heteropolymer of a number of monmeric precursors, which results from a 
biosynthetic pathway in which the monooxygenase catalyzed product of 
tyrosine, DOPA, is polymerized to form the melanin pigment. To obtain 
pigment and the related metabolic products for purification in accordance 
with this method, particularly, melanin, pheomelanin, the precursor DOPA, 
tyrosine, tyrosinase and other related enzymes, bacterial cells are grown 
to the late logarithmic phase in a growth medium after which the 
supernatant containing pigment is collected and filtered. The resulting 
free filtrates are concentrated, for example, by evaporation and dialysis. 
Alternatively, pigments can be precipitated with about 1% potassium 
persulfate and about one volume of methanol and recovered, for example, by 
centrifugation. The collected samples are subsequently deproteinized, for 
example, by extraction with 0.4M perchloric followed by centrifugation. 
Further purification can be facilitated by conventional means such as, for 
example, column chromatography or ion-exchange chromatography to resolve 
fractions. The pigment fractions are subsequently identified, for example, 
by an Absorbance/Fluorescence monitor. Ultrapure tyrosinase products are 
obtained and characterized by high pressure liquid chromatography (HPLC). 
Alternatively, these shellfish inducers are ultrapurified for maximum 
activity by isoelectric focusing or electrophoresis. 
In a further embodiment of this invention, an acid polysaccharide 
exopolymer which is metabolically produced by the present bacteria is 
isolated, for example, by solubilizing the polysaccharide in an 
acetone-alcohol solution. The exopolymer can be precipitated at 
water-solvent interface. 
For a better understanding of the present invention with other and further 
objects, reference is made to the following experimental descriptions and 
examples. 
EXAMPLES 
MATERIALS AND METHODS 
Organism and Culture Conditions 
LST was isolated on Marine Agar (Difco 2216) slants. Cultures were grown in 
a gyratory water bath (New Brunswick Scientific Model G76), at a setting 
yielding 8.5 ppm dissolved oxygen, at 25.degree. C. The media employed 
were Marine Broth (Difco 2216), AGMS Synthetic Medium and AG Synthetic 
Medium, formulated similarly to the AGMS but lacking methionine and 
serine. The exact composition of AGMS and AG broths is set forth below. 
Solid synthetic media were prepared by adding 1.5% Agar (Difco). LST did 
not grow on TCBS. 
COMPOSITION OF THE AGMS AND AG SYNTHETIC MEDIA 
The AGMS Synthetic Medium consists of two stock solutions: 
______________________________________ 
Stock #1: 
______________________________________ 
NaCl 19.45 g/L 
Mg.Cl.sub.2.6H.sub.2 O 
8.80 g/L 
Na.sub.2 SO.sub.4 3.14 g/L 
CaCl.sub.2 (anhydrous) 
1.80 g/L 
KCl 0.55 g/L 
NaHCO.sub.3 0.16 g/L 
KBr 0.08 g/L 
H.sub.3 BO.sub.3 0.022 g/L 
SrCl.sub.2 0.034 g/L 
NaSiO.sub.3 0.004 g/L 
NH.sub.4 NO.sub.3 0.0016 g/L 
Na.sub.2 HPO.sub.4 0.008 g/L 
Ferric Ammonium Citrate 
0.10 g/L 
______________________________________ 
The salts solution is autoclaved at 121.degree. C. for 15 min at 15 lbs 
pressure. Sterile solution was stirred to evenly distribute the 
precipitate formed. 
______________________________________ 
Stock #2: 
______________________________________ 
Aspartic Acid 26.27 g/L 
Glutamic Acid 23.77 g/L 
Methionine 0.39 g/L 
Serine 17.16 g/L 
______________________________________ 
The pH of the Stock #2 solution was adjusted to 7.2-7.4 using 6N NaOH. 
Sterilization by autoclaving as above followed pH adjustment. 
AGMS Medium consists of a mixture of 30 ml Stock #1 with 70 ml Stock #2. 
AG Medium uses the amino acid pool given below: 
______________________________________ 
Stock #3: 
______________________________________ 
Aspartic Acid 26.27 g/L 
Glutamic Acid 23.77 g/L 
______________________________________ 
Stock #1 and Stock #3 were mixed in the same proportions as for AGMS Medium 
(30-70) after adjusting the pH of the solution to 7.6 with 6N NaOH and 
sterilizing the solution. 
______________________________________ 
Stock #4: Phosphate Solution 
______________________________________ 
K.sub.2 HPO.sub.4 13.6 g/L 
K.sub.2 HPO.sub.4 21.3 g/L 
______________________________________ 
Autoclave separately, add 0.46 ml/100 ml GAMS. 
Synthetic Medium Development and Growth Curves 
The AGMS Synthetic Broth sustained the growth of LST when supplemented with 
2% NaCl (NaCl final concentration 3%). Using a drop-out series experiment, 
the contribution of each amino acid supplied in AGMS (aspartic acid, 
glutamic acid, methionine and serine) to the growth of LST was evaluated 
by direct microscopic counts (phase contrast 0.19 .mu.m resolution) and by 
viable counts. 
To approximate the growth rate of LST, turbidimetric measurements of 
cultures grown in Marine and AG broths were made over a period of 470 hrs 
using a Klett-Summerson Photoelectric Colorimeter with a green filter. 
Morphology 
Cell morphology during the growth cycle of LST was monitored under phase 
contrast microscopy (Series 10 AO Microscope 0.19 .mu.m resolution). 
Scanning electron microscopy was used for a more detailed view of the 
structure of normal and aberrant LST cells. Bacterial cells were fixed 
according to a procedure described by Belas and Colwell (1982). To 
minimize the amount of inorganic precipitate, LST cells were grown for 
48-96 hrs in AG Broth. The cultures were then centrifuged (Model PR-G IEC 
Refrigerated Centrifuge) at 2500.times.g for 10 min decanted, resuspended 
in 10 ml PBS and washed twice. After the final centrifugation, the pellets 
were resuspended in 10 ml PBS and 1 ml of 25% glutaraldehyde 
(Polysciences) was added. The mixtures were allowed to fix for 1 hr either 
at room temperature or overnight at 4.degree. C. Following fixation, the 
bacterial suspension was passed through a 13 mm Swinex holder with a 0.2 
.mu.m Nucleopore filter, using a syringe attached to the Swinex. The 
volume that passed through each filter varied between 1 and 5 ml of 
culture suspension; care was taken to avoid damaging both fragile cell 
appendages and the filter. The syringe was then refilled with 5 ml of 0.2M 
cacodylate buffer with 2.5% glutaraldehyde; half the mixture was pushed 
through the filter, and the Swinex holder was sealed and stored overnight 
at 4.degree. C. After fixation, dehydration was accomplished in seven 
steps, in which 5 ml EtOH (sequential concentrations of 30, 50, 70, 90 and 
3.times.100%) were slowly passed through the filter over a period of 30-60 
minutes. 
Specimens were further prepared for microscopy as follows: The filters were 
critical point dried and placed cell side up on SEM stubs using double 
stick adhesives. To reduce charging of the specimen, small drops of silver 
paint were placed on four corners of the stub connecting the filter 
surface to the stub metal. The stubs were coated with Ag/Pd metal alloy in 
a sputter coater, and then stored for scanning electron microscopy in a 
dessicated environment. 
To determine the presence and location of flagella on LST, the procedure of 
Mayfield and Innis (1977), a modification of Gray's stain, was used on wet 
mounts of motile bacteria. Stained cells were examined with phase contrast 
microscopy. 
As anticipated, LST incurred a lengthy lag period (27 hrs) when transferred 
from Marine to AG medium. This lag period was not observed when cultures 
were transferred from AG to AG medium (FIG. 2). The generation time of LST 
at 25.degree. C. was 4 hrs in marine broth and 7 hrs in AG Medium. The 
slower growth rate in the Synthetic Medium is presumably correlated to the 
availability of nutrients in the two media. All growth factors must have 
been synthesized de novo from glutamic and aspartic acids in AG, whereas 
Marine Broth was replete with numerous vitamins from Yeast extract and a 
wide variety of nutrients in peptone. 
Mutagenesis 
Ethyl Methane Sulfonate 
To test the hypothesis that LST pigment attracts spat, pigment-less 
variants were desirable controls Consequently, LST was mutagenized with 
ethane methane sulfonate (EMS; Sigma) according to a modification of the 
procedure used by McCardell (1979). Logarithmically growing cultures of 
LST were suspended in 0.066M PBS to an approximate concentration of 
2.times.10.sup.9 cells/ml. EMS was added to 1 ml aliquots of culture to 
yield final concentrations ranging between 10-30 .mu.l/ml (5 .mu.l 
intervals). The resulting suspensions were incubated for 1 and 1.5 hrs in 
a G76 Water Bath Shaker (New Brunswick Scientific) at setting 5. The 
suspensions were diluted 1:10 in PBS, centrifuged, washed with 5 ml PBS 
and resuspended in 3 ml PBS. Two ml of the final suspension were 
inoculated in AG Broth and incubated 2-5 days. After this adaptation 
period, the mutagenized and recovered culture was then spread on Marine 
Agar. The remaining 1 ml of treated suspension was used to "spread plate" 
directly on AG and Marine agars. Screening of mutants was assessed 
visually, since pigment production was easily scored on agar plates. 
Mutagenesis with EMS for 1 hr reduced the viability of LST 2-3 logs as 
determined by spread plate counts on Marine Agar (Table 6). No colonies 
formed on AG Agar when LST was "plated" directly after mutagenesis. This 
result was not unexpected: Since the minimal medium lacks so many growth 
factors, auxotrophic mutations would be conditionally lethal. Mutagenzied 
suspensions, after 2-5 days "holding" periods in AG Broth, were streaked 
on Marine and AG agars. Spread plate counts on Marine Agar ranged from 
1.3-6.6.times.10.sup.9, while they were approximately two logs lower on AG 
Agar: 1.4-6.5.times.10.sup.7 (Table 6). The colonies on AG Agar were 
probably in part progeny of cells that remained in stasis in the AG 
Medium, repairing damage to the chromosome and possibly even back 
mutating. 
Suspensions treated with EMS (all concentrations) for 1.5 hrs did not yield 
any colonies either after direct plating (Marine or AG agars), or after 
the holding period in AG Broth. 
No pigment-less mutants were detected among the approximately 5000 colonies 
screened, on either undefined or minimal media. A number of factors may 
have led to this failure. Two of the possibilities, not mutually 
exclusive, are that pigment production is part of an obligate cell 
survival pathway, a serious consideration since melanin is part of the 
tyrosinase metabolism. In this case, obtaining pigment-less variants may 
prove an unrealistic goal. A second possibility is based on reports that 
pigment synthesis is essentially dependent upon a single enzyme, 
tyrosinase or a tyrosinase-like derivative. In this instance, mutations 
involving the mel gene would appear with very low frequencies. 
Furthermore, the likelihood of another mutational aberration that would be 
lethal to cells containing a lesion in a mel gene would be high. In any 
case, we had only screened about 5000 colonies by this procedure, and a 
mutation rate of less than 0.02% is not uncommon. Mutagenesis experiments 
using ICR 191 were designed with a holding period in Marine Broth rather 
than AG Broth to minimize auxotrophic lethality. 
ICR 191 
The procedures were modified slightly from those described above. The 
reaction mixture consisted of AG minimal medium containing 10% Marine 
Broth, 3-6.times.10.sup.8 LST/ml and 10 .mu.g ICR 191/ml. Cells were 
incubated at 30.degree. C. in the reaction mixture for 12 hrs and then 
diluted 1:100 into fresh Marine Broth to provide an adaption period of 
between 12-72 hrs. Mutants were screened on Marine, AG and AGT agars. 
A total of 24,803 colonies were screened. Thirty-nine colonies varied in 
pigmentation, seven had no pigment (hypo), two were darker (hyper), 
including the HYP variant, two were light tan, 27 were various shades of 
red from which the DIF variant was isolated and one was yellow. The 
paucity of pigment mutations suggested that either only a single enzyme 
was necessary for pigmentation (or any one of two or more enzymes) or that 
somehow pigmentation was somehow linked to viability. The first of these 
two hypotheses is consistent with the pigment being a melanin. These 
results also suggest that LST may produce more than one pigment, the 
lighter ones being masked by the brown ones. Also interesting, the seven 
mel- or hypo "mutations" have not been stable, reverting on average about 
one in 3-10 generations. 
About 83% of the colonies that grew in Marine Agar, grew on AG Agar 
revealing that a considerable fraction of auxotrophic mutations were 
produced. Inexplicably, only 66% of the colonies that grew on Marine Agar, 
grew on AGT Agar. 
Pigment Isolation and Characterization 
Crude pigment was obtained from broth cultures that had been grown for at 
least 48-72 hrs (to stationary phase) in either Marine or AG broths. Spent 
medium was centrifuged at 2500.times.g for 15 min to remove the cells. The 
supernatants were dialyzed against distilled water for 24 hrs and pigment 
was purified by gel filtration. 
Sephadex G-50, G-75 and G-150 columns (Pharmacia Chemicals), in which the 
dextran beads were swollen in distilled water in 0.02% sodium azide to 
prevent microbial growth, were calibrated with lysozyme, tripsinogen, egg 
albumin, bovine albumin and yeast alcohol dehydrogenase standards obtained 
from Pharmacia. Running buffer consisted of distilled water with 0.02% 
sodium azide, adjusted to pH 8.5. Void volume was determined using blue 
dextran 2000. The fractions were monitored at 280 nm. 
The pigment fractionation was carried out on an Isco Model 328 Fraction 
Collector, using an ISCO Type 6 Optical Unit and an ISCO Model UA-5 
Absorbance/Fluorescence Monitor to identify the pigment fractions. 
The optical densities of the Sephadex fractions were analyzed using a Model 
25 Beckman Spectrophotometer in the scan mode (200 through 750 nm). In 
general, melanin had a much lower extinction coeffecient in the visible 
range than in the ultraviolet, making dilutions of the samples necessary 
for analysis in the range of 200-350 nm. The absorption spectra of 
glutaraldehyde-treated cultures were also determined. Using a second basic 
method of extraction, crystallized pigment was obtained by a procedure in 
which the liquid phase of a culture supernatant was boiled off and the 
"residue" was dried at 90.degree. C. 
Another experiment was designed to determine whether a significant amount 
of pigment was cell-associated, or whether most of the pigment was 
excreted. Cell pellets (2500.times.g, 15 min) were resuspended in 
phosphate buffered saline (PBS), sonicated at low speed (setting 30) for 
30 seconds (Bronwill Biosonk IV Sonicator) and recentrifuged. This 
pigment preparation was compared spectrophotometrically to a culture, 
containing both cells and soluble pigment, treated in the same way with 
sonication. Standard solutions of melanin (Sigma) at a concentration of 
0.25 mg/ml distilled H.sub.2 O and L-DOPA (Sigma) at a concentration 1.0 
mg/ml were compared with the absorbance spectra of LST culture pigments. 
Pigment solubilities were preliminarily tested, using 0.5 ml culture 
supernatant to 2.5 ml solvents. The solutions were agitated and maintained 
for at least 30 min after which they were centrifuged to separate 
potential precipitates. The criteria of Zussman, et al. (1960) were 
adopted to describe the solubility of pigment in the solvents. Pigments 
were designated "soluble" if they dissolved in the solvent, "slightly 
soluble" if the solvent became colored but the pigment did not dissolve, 
and "insoluble" if no color was imparted to the solvent. Solvent-pigment 
combinations were also examined by spectrophotometer. 
Infrared (IR) spectra were determined (Perkin Elmer 281 IR 
spectrophotometer). Experimental samples were column purified, dialyzed, 
freeze dried LST pigment from culture supernatant to which one drop of 
paraffin oil was added. Commercially obtained melanin (Sigma), synthesized 
via the photooxidation of L-DOPA, was used as a control. 
After LST cultures reached stationary phase, a soluble pigment, ranging in 
color from reddish-brown to dark brown, became evident. It was retained in 
dialysis and was precipitated by acidified water, ethanol and methanol 
(Table 4). The pigment was relatively soluble in water, only slightly 
soluble in ethanol and methanol and insoluble in acetone, chloroform, 
cyclohexane and ethylene dichloride. 
The crude pigment exhibited three maximum absorbance intervals at 260, 407 
and the largest at 220 nm (Table 5, FIG. 3). Glutaraldehyde partially 
oxidized the pigment, shifting the absorbance peaks to 233, 273 and 435 
nm. When the pigment was totally oxidized, it appeared darkest and an 
absorbance peak was shifted still further from 273 to 293 nm. 
Additionally, there was generalized absorption in the visible region. 
The absorption spectra of the experimental LST pigment was compared with 
spectra of commercial melanin, which had peaks at 225 and 273 nm, and with 
L-DOPA, which had peaks at 233, 282 and 512 nm. The LST product absorbance 
maxima were deemed to sufficiently match those of the commercial 
preparation to conclude that LST did indeed produce a melanin. Further 
purified LST pigments tended to support that hypothesis. Pigment fractions 
obtained from Sephadex G-75 and G-150 columns yielded absorbance maxima at 
226, 263 and 407 nm (FIG. 4). A peak in the visible region was not 
detected in the commercial pigment preparation, possibly due to the 
consequence of the low solubility of melanin (viz., a particle-free 
suspension was not sufficiently concentrated). 
Shellfish Attachment 
Three spat setting tanks were filled with seawater (25.degree. C.) and 
presetting (eyed) oyster larvae. Acid cleaned (1N HCl, 24 hrs) and 
sterilized glass slides were immersed in Set Tank 1. Glass slides, 
categorized and treated as follows, were placed in Set Tank 2: 
1. Pigmented LST: Slides were immersed for 24 hrs in a stationary phase 
culture of LST, grown in Marine Broth at 25.degree. C. 
2. UV irradiated LST: Slides were immersed for 24 hrs in a late stationary 
phase culture of LST, grown in Marine Broth at 25.degree. C. The slides 
were then exposed to lethal doses of UV radiation. 
3. Marine Broth control: Slides were immersed in uninoculated media for 24 
hrs. 
In Set Tank 3, plates and glass slides were treated in the following 
manner: 
1. 10 mg DOPA per 10 ml 2% noble agar. 
2. 50 mg DOPA per 10 ml 2% noble agar. 
3. 100 mg DOPA per 10 ml 2% noble agar. 
4. 10 mg commercial melanin per 10 ml 2% noble agar. 
5. 20 mg commercial melanin per 10 ml 2% noble agar. 
6. 10 ml noble agar (control). 
7. Culture pigment: An LST culture in the late stationary phase of growth 
(Marine Broth at 25.degree. C.) was centrifuged (2500.times.g, 10 min) and 
the supernatant filtered through 1.2 .mu.ml Millipore filters to further 
remove cells. Slides were immersed in the cell-free filtrate for 24 hrs. 
After 24 hrs in the setting tanks, all slides and plates were removed and 
the attached spat were counted using a stereoscope (10X; Baush and Lomb). 
One caveat must be noted. The pigment coated slides and all of the Agar 
plates were placed in one tank. The DOPA dissolved in the water (high 
solubility, large water volume), autooxidized, and a thin deposit coated 
all the plates, slides and tank surfaces. Thus, the attached spat 
population may have been enhanced. 
Data such as those reported in Tables 7 and 8, together with other 
evidence, supports the notion that LST pigment promotes shellfish 
attachment. Slides coated with pigmented LST attracted more than 5 times 
the oyster spat than the clean and control slides (Tables 7 and 8). 
Interestingly, slides coated with UV-irradiated LST attracted slightly 
less spat than the controls, possibly because the melanin was 
photooxidatively degraded. 
The data involving Agar plate imbedded with the test substance and glass 
slides coated with culture pigment are to be interpreted much more 
cautiously, since the DOPA diffused out of the Agar, autooxidized, 
infiltrated the tank and interfered with experimental gradients. 
Nevertheless, melanin Agar plates also attracted more spat than the 
control plates, while DOPA Agar plates attracted 2-5 times less spat than 
the controls. The number of spat (attached oysters) was inversely 
proportional to the concentration of DOPA in the Agar, suggesting that at 
high concentrations, DOPA may have a repelling effect on the shellfish. 
The pigment coated slides, placed in the same tank with the Agar plates, 
attracted almost 10 times the number of spat attached to the control 
slides. 
Isolation and Purification of Acid Polysaccharide Exopolymer 
LST, DIF and HYP in 3% brain heart infusion agar (3 BHI) secretes an acid 
polysaccharide exopolymer amounting to approximately 500% of the weight of 
the bacterium in 48 hrs at 25.degree. C. This exopolymer is isolated as 
follows: 
1. 3 BHI in large petri plates is inoculated with 1 ml of 10.sup.8 LST, DIF 
or HYP grown as described above. 
2. The cultrue is incubated for 2 days. 
3. The acid polysaccharide exopolymer is solubilized in an acetone-alcohol 
solution. 
4. The exopolymer is precipitated at the water-solvent interface. 
Larval Induction 
LST, DIF and HYP are grown in a conventional growth vessel fermentator (NBC 
CMF 128S) filled to 15 liters. A series of these vessels accomodates 
coated cultch or slides to induce the metamorphosis of 10,000 spat. 
The bacterial cells are grown as indicated above and slides are immersed in 
the growth vessel. The bacteria affix to the slides via the adhesive 
exopolymer they produce. 
The bacteria-coated surfaces are removed from the fermentor and placed at 
the bottom of a larval set tank. Larvae are exposed to the bacteria in 
filtered, slow moving water. The steady state setup can remain active for 
months. The activated cultch or slides are restored each new setting 
season Salinities range from 1.8 to 2.5%. 
The oysters are fed Isochrysis galbana and Monochrysis lutheri at a rate of 
approximately 2.times.10.sup.5 cells/ml of culture per day. 
Thus, while the invention has been described with reference to certain 
preferred embodiments, those skilled in the art will realize that changes 
and modifications may be made thereto without departing from the full and 
intended scope of the appended claims. 
TABLE 1 
______________________________________ 
Some biochemical and physical 
characteristics of LST 
Test Reaction 
______________________________________ 
Gram stain gram neg 
Cell shape rod 
Spores - 
Motility + 
Catalase + 
Lysine decarboxylase 
+ 
Ornithine decarboxylase 
- 
Sucrose fermentation 
- 
Mannose fermentation 
- 
Arabinose fermentation 
- 
Growth in 2.5% NaCl 
+ 
Growth in 5.0% NaCl 
+ 
Growth in 7.5% NaCl 
+ 
______________________________________ 
TABLE 2 
______________________________________ 
Contributions of aspartic acid (asp), 
glutamic acid (Glu), methionine (Met) and 
serine (Ser) to the growth of LST.sup.a 
Amino Acids Growth.sup.b 
______________________________________ 
Asp Glu Met +++ 
Asp Met Ser +++ 
Glu Met Ser +++ 
Asp Ser +++ 
Ser Met - 
Asp Glu +++ 
Asp Met + 
Ser Glu ++ 
Glu Met ++ 
______________________________________ 
.sup.a Inorganic salts solution (Appendix I) was supplemented with each o 
the amino acids listed in the concentrations used in the AGMS medium. 
.sup.b +++, .about.7 hr generation time; ++, .about.10 hr generation time 
+, .about.13 hr generation time; -, no growth. 
TABLE 3 
__________________________________________________________________________ 
Adenosyl nucleotide pool in a hypo-pigment producing 
varient of LST cultivated in batch culture.sup.a 
Growth Viable pM 
Phase Count (cfu/ml) 
Morphology 
AEC.sup.b 
ATP/Cell 
.mu.g/Cell.sup.c 
__________________________________________________________________________ 
Log 4.5 .times. 10.sup.7 
Short Rods 
0.86 
1.19 .times. 10.sup.-7 
6.56 .times. 10.sup.-11 
Stationary 
2.9 .times. 10.sup.9 
Rods 0.80 
1.73 .times. 10.sup.-9 
9.53 .times. 10.sup.-13 
Stationary-Decline 
2.7 .times. 10.sup.7 
Long Spirals 
0.72 
1.61 .times. 10.sup.-9 
8.86 .times. 10.sup.-13 
__________________________________________________________________________ 
.sup.a Cells were removed from a batch culture of LST during log phase, 
stationary phase and during the stationarydecline transition and were the 
frozen (-70.degree. C.). Adenoxyl nucleotides were extracted in boiling 
tris. The samples were then assayed for ATP, ADP and AMP and adenylate 
energy charge (AEC) was calculated. 
.sup.b Calculations were based on known internal standards that revealed 
recovery and counting efficiencies if 72.4% for ATP, 41.6% for ADP, 38.0% 
for AMP. 
.sup.c .mu.g ATP per cell was calculated by multiplying pM/cell by 
10.sup.-6 and by ATP mol. wt. 
TABLE 4 
______________________________________ 
Solubility of LST excreted pigment (in 
spent medium) in seven solvents 
Absorbance.sup.c 
Solvent Solubility.sup.a 
Precipitate.sup.b 
Maxima 
______________________________________ 
H.sub.2 O, pH 3 
S +(24 h) 264, 401 
H.sub.2 O, pH 9 
S - 264, 407 
Acetone I +(30 s) none 
Ethanol SS +(30 s) 254, 375 
Methanol SS +(30 s) 246, 264, 400 
Ethylene I - 233, 264, 400 
dichloride 
Chloroform 
I - 243, 276 
Cyclohexane 
I - 203, 222 
______________________________________ 
.sup.a Srelatively soluble; SSslightly soluble; Iinsoluble 
.sup.b + Precipitate formed (time at which formed) - No precipitate forme 
.sup.c Absorbance maxima of pigmentsolvent mixtures vs. solvent reference 
TABLE 5 
______________________________________ 
Spectral absorbances of pigments 
extracted from LST 
Absorbance 
Optical densities 
Sample Dilution Maxima.sup.a 
at Abs. Maxima 
______________________________________ 
Marine Broth 1:8 260 1.10 
Supernatant 1:4 407 0.50 
LST-Associated 
1:16 264 0.36 
Pigment 1:4 407 0.33 
LST-Associated and 
1:64 260 0.38 
Soluble Pigment 
1:4 407 0.25 
Red-Black Pigment 
1:64 237 1.08 
Treated w/Glut.sup.b 
1:1000 293 0.24 
Dark Orange Pigment 
1:64 234 1.34 
Glut- 1:64 272 0.58 
1:4 436 0.60 
Yellow Pigment 
1:64 232 1.17 
Glut- 1:64 274 0.38 
1:4 434 0.22 
Orange Pigment 
1:64 265 2.00 
Glut- 1:2 436 0.34 
Crude Pigment 
1:4 256 1.16 
Extract.sup.c 
none 405 
0.11 
Commercial Melanin 
1:4 225 0.43 
0.25 mg/ml 1:4 273 0.31 
Commercial L-DOPA 
1:4 233 3.00 
1.0 mg/ml 1:4 282 2.90 
none 512 0.37 
______________________________________ 
.sup.a There were 2-3 maxima for each sample. See text and FIG. 3 legend 
for further detail. 
.sup.b Gluteraldehyde, an SEM fixative. 
.sup.c Crude pigment extract was obtained by redissolving crude pigment 
crystals in distilled water to solubility limit (exact concentration 
unknown). 
TABLE 6 
______________________________________ 
Toxicity of Ethyl Methane Sulfonate 
(EMS) to LST.sup.a 
EMS Conc. 
Direct Growth.sup.b 
Growth after Holding.sup.c 
ug/ml MA AG MA AG 
______________________________________ 
10 3.7 .times. 10.sup.6 
No Data.sup.d 
1.4 .times. 10.sup.9 
1.5 .times. 10.sup.7 
15 1.3 .times. 10.sup.6 
" No Data No Data 
20 7.0 .times. 10.sup.7 
" 6.6 .times. 10.sup.9 
6.5 .times. 10.sup.7 
25 6.9 .times. 10.sup.7 
" 3.2 .times. 10.sup.9 
2.1 .times. 10.sup.7 
30 7.1 .times. 10.sup.7 
" 1.3 .times. 10.sup.9 
1.4 .times. 10.sup.7 
original 6.7 .times. 10.sup.9 
3.8 .times. 10.sup.9 
-- -- 
culture 
______________________________________ 
.sup.a LST was exposed to EMS concentrations for 1 hr. 
.sup.b Mutagenized suspensions were spread on plates immediately after 
exposure to EMS. 
.sup.c Aliquots of mutagenized suspensions were "held" in AG Broth for 2- 
days, after which they were spread on Marine (MA) and AG agars. 
.sup.d The dilutions plated did not yield any colonies. 
TABLE 7 
______________________________________ 
Density of Crassostrea virginica larvae 
attached to glass and agar surfaces 
Slide or Agar Plate Attached Spat 
Preparation.sup.a Density.sup.b 
______________________________________ 
Clean and Marine Broth 
0.11/in.sup.2 (16) 
Control Slides (I) 
Pigmented LST (II) 0.58/in.sup.2 (16) 
UV Irradiated LST (II) 
0.07/in.sup.2 (16) 
Culture Pigment (III) 
1.00/in.sup.2 (10) 
10 mg DOPA/10 ml agar (III) 
1.03/in.sup.2 (2) 
50 mg DOPA/10 ml agar (III) 
0.42/in.sup.2 (2) 
100 mg DOPA/10 ml agar (III) 
0.42/in.sup.2 (2) 
10 mg melanin/10 ml agar (III) 
2.22/in.sup.2 (2) 
20 mg melanin/10 ml agar (III) 
2.55/in.sup.2 (2) 
Agar Control/10 ml agar (III) 
2.12/in.sup.2 (2) 
______________________________________ 
.sup.a Number in parentheses designates the spat tank used. 
.sup.b Number in parentheses designates the number of samples taken. 
TABLE 8 
______________________________________ 
Attraction of Crassostrea virginica larvae by the 
bacteria LST, a melanin synthesizing species 
Sample Mean No. 95% Confidence 
Sample Type.sup.a 
No..sup.b 
Spat at 24 h.sup.c 
Interval.sup.d 
______________________________________ 
Control.sup.e 
16 3.8 .+-. 1 1&lt; &gt;7 
Prefouled.sup.f 
27 24.8 .+-. 7 
12&lt; &gt;38* 
Hyphomonas 14 5.0 .+-. 2 2&lt; &gt;8 
neptunium.sup.g 
LST.sup.g 16 17.5 .+-. 3 
12&lt; &gt;23* 
Pigment from LST.sup.g 
10 30.0 .+-. 14 
2&lt; &gt;58 
UV Killed LST.sup.g 
16 1.8 .+-. 1 0&lt; &gt;4 
Control.sup.e 
5 2.4 .+-. 1 2&lt; &gt;3 
LST.sup.g 14 9.7 .+-. 1 7&lt; &gt;12* 
LST Hypopigment 
8 2.0 .+-. 1 1&lt; &gt;4 
producer.sup.g 
______________________________________ 
.sup.a First 6 samples were run on 1/81 and last 3 samples were run on 
7/81 at the oyster mariculture unit in Lewes, Delaware. 
.sup.b Chemically cleaned and sterilized 3 in .times. 1 microscope slides 
.sup.c Larval settlement and/or attachment per slide .times. 10. Standard 
error also shown. 
.sup.d Asterisk denotes significant deviation from control samples. 
.sup.e Placed in Marine medium for 24 hrs prior to immersion in oyster 
tank. 
.sup.f Slides placed in mariculture holding tank (.about.10.sup.5 viable 
bacteria/ml) prior to immersion in oyster tank. 
.sup.g Slides were coated with sample type prior to immersion in oyster 
tank. 
TABLE 9 
__________________________________________________________________________ 
LST properties of pigment compared with pigments 
identified as melanin of other microorganisms 
ORGANISMS 
Aeromonas.sup.a 
Vibrio.sup.b 
Aspergillus.sup.c 
PROPERTIES liquefaciens 
cholerae 
nidulans 
LST.sup.d 
__________________________________________________________________________ 
Color Brown-Black 
Brown 
Black Brown 
Solubility in H.sub.2 O at pH 7 
I ND ND SS 
Solubility in 0.1 N NaOH 
S S S S 
Blackberg-Wanger Precipitation 
PPT PPT ND PPT 
FeCl.sub.3 Precipitation 
PPT PPT PPT PPT 
Acid Precipitation 
PPT PPT PPT PPT 
Reduction (Glutathione) 
+ + + + 
Reoxidation + ND + + 
Absorption Peaks 
Diffuse 
345,480 
480,535 
264,407 
H.sub.2 O Bleaching 
ND + + + 
Molecular Weight 
ND ND 2,000,000 
120,000 
350,000 
52,000 
29,000 
12,000 
__________________________________________________________________________ 
I-insoluble; Ssoluble; NDno data; PPTprecipitated; positive 
.sup.a Aurstad and Dahle 1972 
.sup.b Ivins and Holmes 1980 
.sup.c Bull 1970 
.sup.d Present study