MDU-T-82-005 C. 3

Induction of SettlementAnd MetamorphosisIn Crassostrea viroinicaBy a Melanin-SynthesizingBacterium

Ronald M. WeinerRita R. Colwell

CffiCULATM COPYSfi8 firant Depository

Technical ReportMaryland Sea Grant Program1224 HJ. Patterson Hal!College Park, Maryland 20742

vi Publication NumberS OM-SQ-TS-82-05

"♦OB*" NATIOSW SEA GJWIT DEPOSITORYPFLL LiUVi^V iVjlLU'.fiCi

URI HARKftGAi^tTT SAV CAMPUS'nARRAGAHSETT, Rl 02S82

rYYDuL-T"- %Zk~OOB C3

INDUCTION OF SETTLEMENT AND METAMORPHOSIS

IN CRASSOSTREA VIRGINICA

BY A MELANIN-SYNTHESIZING BACTERIUM

Project Directors

Ronald M. Weiner

Department of MicrobiologyUniversity of MarylandCollege Park, Maryland

Rita R. ColwellDepartment of Microbiology

University of MarylandCollege Park, Maryland

Copies of this publication are available from:

Sea Grant ProgramUniversity of Maryland122* H J. Patterson Hall

College Park, MD 207*2

UM-SG-TS-82-05

The publication of this report is made possible by grant//NA81AA-D-000*0, awarded by the National Oceanic and Atmospheric Administration to the University of Maryland SeaGrant Program.

The University of Maryland is an equal opportunity institutionwith respect to both education and employment. The University's policies, programs and activities are in conformancewith pertinent federal and state laws and regulations on nondiscrimination regarding race, color, religion, age, nationalorigin, sex and handicap. The University complies with TitleVI of the Civil Rights Act of 196*, as amended, Title IX ofthe 1972 Education Amendments and Section 50* of the Rehabilitation Act of 1973.

TABLE OF CONTENTS

Editor's Preface 1

Introduction 3

Background 5

Materials and Methods 8Organism and Culture

Conditions 8Synthetic Medium Development

and Growth Curves 8Morphology 8Pigment Isolation and

Characterization 10Mutagenesis 11Shellfish Attachment 12

Results [if.Formulation of a

Synthetic Medium I*Morphology and Morphogenesis 1*Pigment Characterization 15Mutagenesis 16Shellfish Attachment 18

Summary 19

Tables 21

Figures 3[

Literature Cited 39

Appendix 43

111

EDITOR'S PREFACE

The oyster, of all invertebrate organisms, has been perhaps the most vigorously studied. A recent Maryland SeaGrant survey of oyster literature (Breisch and Kennedy, A_ Selected Bibliography of Worldwide Oyster Literature, 1981)lists almost *000 articles, books and theses covering a rangeof issues: from oyster biology, reproduction and genetics tolarval behavior to resource management and aquaculture. Asstudy of oyster biology progresses and as laboratory technology becomes more sophisticated, our understanding of thefundamental mechanisms of larval recruitment has increasedsignificantly. One of those mechanisms is the subject of thisreport: the biochemical processes that are involved in thesettlement of larvae and the consequent metamorphosis toyoung oysters, or spat.

Previous research by Ronald Weiner and Rita Colwellidentified specific microbial communities on oyster cultchthat hinder or help settlement. Here, they present evidencethat larvae are attracted to surfaces by the melanin pigmentof a newly observed bacterium species they call LST. Thepigment is the product or precursor of LST. This studyfocuses primarily on the life cycle of LST, particularly thebiochemical pathways of pigment formation and pigmentidentification.

Free swimming larvae, shortly after spawning, seek asuitable place to settle and attach themselves. A number ofenvironmental conditions are involved in settlement—salinityand nutritional availability are probably the most important.But once larvae are satisfied with those conditions, they appear to respond to a biochemical cue to settle and attachthemselves. That biochemical cue is a pigmented bacteriumwhich adheres strongly to surfaces like oyster shell. In anumber of controlled experiments using different bacterialcoatings, with and without the melanin pigment, Weiner andColwell found that more larvae settled on the pigment-coatedsurfaces than any other.

There may be a symbiotic relationship between bacteriaand oyster larvae: Electron microscopy experiments show

that the LST bacterium undergoes various morphogenicchanges, leading finally to terminal growth. Settlement by.larvae may provide bacteria with nutrients that inhibit oreven reverse this process; or it could be that larvae stimulatebacterial reproduction. While these explanations of evolutionary adaptation remain to be investigated, the more immediate implications of a bacterial-oyster larvae symbiosis maybe that some shellfish-borne diseases are the result of natur

ally occuring biological interactions, a finding that could alter current hygienic guidelines.

Over the long term, the study of the mechanisms of larval attraction could lead to the development of a safe chemical attractant capable of inducing spat set: this could significantly improve the productivity of seed hatcheries or, on natural sites, cultch could be treated prior to returning it tooyster bars.

Merrill Leffler

INTRODUCTION

A marine bacterium, designated LST, has been isolatedand appears to have a specific, beneficial role in the settingand metamorphosis of Crassostrea virginica larvae. LST is anaerobic, gram-negative rod, highly motile with an overall deoxyribonucleic acid (DNA) base composition of *5.6% guano-sine and cytosine. It grows optimally in a 3.5% (w/v) marinesalts mixture at 25°C. The bacteria readily attach to a variety of surfaces, including glass, plastic, aluminum and oystershell. Based on its biochemical characteristics and its morphology—it becomes tightly coiled in one phase of growth—wehave concluded that the bacterium is a new species.

When grown in Marine Broth (2216), under optimum conditions of temperature (25°C) and pH (7.3), LST demonstratesa generation time of 70 min. In a synthetic medium consisting of 3.5% marine salts plus 125mM aspartic acid and glutamic acid, the maximum generation time is *00 min. In stationary and decline phases of growth, correlated with a decrease in energy charge (i.e., ATP + 1/2 ADP/ATP + ADP +AMP) from 0.86 to 0.72;LST synthesizes and releases a brownpigment. Based on chemical properties and UV absorbancemaxima, the pigment has been identified as melanin. Whenpurified in a column containing Sephadex 150, it comprisespolymers of various molecular weights, ranging from 12,000-120,000 Daltons, typical of melanin pigments. Assays for LSTtyrosinases are weakly positive.

To verify the hypothesis that LSTand its product/precursor of melanin synthesis attract oyster larvae, microscopeslides were coated with either (a) bacteriological growth medium (control), (b) a mixed culture, including LST, (c) a non-pigmented marine bacterium, Hyphomicrobium neptunium, (d)LST, (e)LST pigment, (f) killed LST, (g) pigment-less LST mutants and (h) LST hyperpigment-producers. The slides wereimmersed in tanks containing 2-5 eyed larvae/ml in sand-filtered, estuarine water collected from Indian River inlet, Delaware.

Prefouled, LST-coated and pigment-coated slides attracted larger numbers of larvae over a 2*-hour interval than control slides. Furthermore, the hyperpigment-producing variantattracted more larvae than the prefouled or LST-coated samples. Non-pigmented strains of H. neptunium and, significantly, pigment-less LST variants attracted neither more norless larvae than control slides coated with the culture medi

um alone. UV-killed strains of LST appeared to repel larvae.It is possible that altered melanin and/or precursor compounds may be competitive inhibitors of the larval settlementinducer.

The relationship observed between LST and Crassostreavirginica larvae suggests that LST, which adheres verystrongly to cultch and other hard surfaces, forms micro-colonies on cultch. We have hypothesized that when sufficientnumbers of bacteria are achieved, during the decline phase ofgrowth, the bacterial colonies produce a high concentrationof pigment, sufficient to attract oyster larvae. The larvaemay be able to "ingest" (i.e., feed upon) the elongated cells(55 um) of LST, observed to occur at that stage of growth.An oyster product could induce LST reproduction, similar tolectins produced by Halachondrea panicea, which stimulatethe bacterium Pseudomonas insolita. The association be

tween LST and oyster larvae may involve a hormone-likestimulatory effect on, or function involved in, larval development and metamorphosis.

BACKGROUND

Lewes spat tank isolate (LST) is a marine organism originally isolated (A. Zachary, personal communication 1978) atthe University of Delaware Mariculture Laboratories, Lewes,Delaware. It was found in close association with oyster spatattached to glass slide surfaces.

LST is a Vibrio-like aerobic, gram-negative rod, highlymotile, with a guanosine + cytosine (G+C) ratio of *5.6% (G.Blumberg, personal communication). It grows optimally in 35ppt salt (range 15-100 ppt) at 25°C and does not producespores. It has been observed to attach to a variety of surfaces, preferentially on glass and oyster shells, but also onplastic and aluminum. Table 1 outlines biochemical characteristics (B. Wolff, personal communication).

Attachment of marine bacteria to surfaces is a very common occurrence (Corpe 1970). In an aquatic environment,where nutrient concentrations are very low and nutrients generally accumulate at interfaces, bacteria must often attachto solid surfaces as a prerequisite for growth (ZoBell 1943).Adler (1966) has shown that the attraction of microorganismsto nutient rich surfaces is a chemotactic response. Surfacecomponents such as fimbriae, flagella, stalks, capsules, slimelayers and inorganic cements all have adhesive properties andmay play a role in attachment (Corpe 1970). The resultingformation of microbial films on surfaces, known as microfoul-ing, also promotes the settlement of larvae of marine animals(Crisp and Ryland 1970; Meadows and Williams 1963).

In the stationary phase of growth, LST routinely producesa reddish brown pigment, which we have classified with themelanins; the pigment appears to mediate the interaction between LST and oyster larvae. It is important to note thatoyster larvae must settle prior to metamorphosis, and may doso in response to chemical cues (Levandowsky and Hauser1978). Larval settling responses triggered by biological compounds have been documented. These include the attachmentof lined chiton larvae on pieces of encrusting corralline algaeor on pieces of ceramic roofing tile soaked in coralline algal

extract (Barnes and Conor 197*) and the induced settlementof barnacle cyprid larvae on slates treated with extracts ofadult barnacles, mussels and oysters (Larman and Gabbott1975). Unpublished reports that DOPA, a melanin precursor,increases oyster spat attachment (S. Coon, personal communication) supports the hypothesis that the bacterial pigmentpromotes the settling of the larvae.

The relationship between LST and the oyster may proveto be a symbiotic one. In the laboratory, LST undergoes morphological changes—spiraling and elongation (Figure I)—leading to a terminal growth stage. These morphogenic eventsoccur after prolonged storage and can be induced by toxiccompounds. A nutrient or other factor provided in situ by theoyster may inhibit or even reverse this terminal stage. A^priori, it would seem that LST might gain survival advantagesas an epiphyte, deriving nutrients from the association withthe oyster. Muller (1981) reported an analogous relationshipbetween bacteria and sponges: In this case, a lectin providedby the sponge was necessary for the growth of the bacteria.

Melanin pigments occur widely in animals and plants,though less often in bacteria. While quite heat stable, theyare highly insoluble and thus difficult to purify. Moreover,enzymes appear to mediate only the first two steps of bacterial melanin synthesis, making the pathway impossible tostudy with enzyme inhibitors and substrate analogs (Nicolaus1968).

The Raper-Mason theory of melanogenesis (Mason 1953;see below) has been refined by subsequent researchers(Nicolaus 1962; Nicolaus and Piatelli 1965; Blois et al. 196*:Swan 196*; Robson and Swan 1966; Kirby and Ogunkoya 1965)who have shown that the melanin macromolecule is actually aheteropolymer of a number of different monomeric precursors. The extent to which the various intermediates are incorporated into the final product and the actual number ofsubunits are believed to vary with the biological system ofsynthesis.

Enzyme EnzymeTyrosine »-DOPA >-Dopaquinone->-Leucodopachrome

Dopachrome—>-5,6-Dihydroxyindole *-5,6-IndoIequinone

>- Melanin

Tyrosinase (EC 1.10.3.1) is the most prominent enzyme involved in melanin synthesis. It is relatively non-specific, exhibiting both cresolase (the hydroxylation of monophenols)and catecholase (the oxidation of o-diphenols) activities.Other melanin related enzymes, including polyphenol oxidases, protyrosinases and heat stable tyrosinases, are found invarious animal, plant and bacterial sources.

The present study focuses on the life cycle of LST, especially pigment formation and pigment identification, and thepotential role of pigment in the attachment of shellfish.

MATERIALS AND METHODS

Organism and Culture Conditions

LST was isolated by A. Zachary and subcultured on Marine Agar (Difco 2216) slants. Cultures were grown in a gyratory water bath (New Brunswick Scientific Model G76), at asetting yielding 8.5 ppm dissolved oxygen, at 25°C. The media employed were Marine Broth (Difco 2216), AGMS Synthetic Medium (Havenner 1976; McCardell 1979) and AG Synthetic Medium, formulated similarly to the AGMS but lackingmethionine and serine. The exact composition of AGMS andAG broths is given in Appendix I. Solid synthetic media wereprepared by adding 1.5% Agar (Difco). LST did not grow onTCBS.

Synthetic Medium Development and Growth Curves

The AGMS Synthetic Broth developed by Havenner (1976)sustained the growth of LST when supplemented with 2%NaCl (NaCl final concentration 3%). Using a drop-out seriesexperiment, the contribution of each amino acid supplied inAGMS (aspartic acid, glutamic acid, methionine and serine) tothe growth of LST was' evaluated by direct microscopiccounts (phase contrast 0.19 urn resolution) and by viablecounts.

To approximate the growth rate of LST, turbidimetricmeasurements of cultures grown in Marine and AG brothswere made over a period of *70 hrs using a Klett-SummersonPhotoelectric Colorimeter with a green filter.

Morphology

Cell morphology during the growth cycle of LST wasmonitored under phase contrast microscopy (Series 10 AO Microscope 0.19 um resolution). Scanning electron microscopywas used for a more detailed view of the structure of normaland aberrant LST cells. Bacterial cells were fixed according

to a procedure described by Belas and Colwell (1982). Tominimize the amount of inorganic precipitate, LST cells weregrown for *8-96 hrs in AG Broth. The cultures were thencentrifuged (Model PR-G IEC Refrigerated Centrifuge) at2500 x g for 10 min. decanted, resuspended in 10 ml PBS andwashed twice. After the final centrifugation, the pelletswere resuspended in 10 ml PBS and 1 ml of 25% glutaralde-hyde (Polysciences) was added. The mixtures were allowed tofix for 1 hr either at room temperature or overnight at *°C.Following fixation, the bacterial suspension was passedthrough a 13 mm Swinex holder with a 0.2 urn Nucleopore filter, using a syringe attached to the Swinex. The volume thatpassed 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% glutaral-dehyde; half the mixture was pushed through the filter, andthe Swinex holder was sealed and stored overnight at *°C.After fixation, dehydration was accomplished in seven steps,in which 5 ml EtOH (sequential concentrations of 30, 50, 70,90 and 3 x 100%) were slowly passed through the filter over aperiod of 30-60 minutes.

Specimens were further prepared for microscopy (P.Ames, University of Maryland) as follows. The filters werecritical point dried and placed cell side up on SEM stubs usingdouble stick adhesives. To reduce charging of the specimen,small drops of silver paint were placed on four corners of thestub connecting the filter surface to the stub metal. Thestubs were coated with Ag/Pd metal alloy in a sputter coater,and then stored for scanning electron microscopy in a dessi-cated environment.

To determine the presence and location of flagella onLST, 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.

Pigment Isolation and Characterization

Crude pigment was obtained from broth cultures that hadbeen grown for at least *8-72 hrs (to stationary phase) ineither Marine or AG broths. Spent medium was centrifugedat 2500 x g for 15 min to remove the cells. The supernatantswere dialyzed against distilled water for 2* hrs and pigmentwas purified by gel filtration.

Sephadex G-50, G-75 and G-150 columns (PharmaciaChemicals), in which the dextran beads were swollen in distilled water with 0.02% sodium azide (Baker) to prevent microbial growth, were calibrated with lysozyme, tripinogen,egg albumin, bovine albumin and yeast alcohol dehydrogenasestandards obtained from Pharmacia. Running buffer consisted of distilled water with 0.02% sodium azide, adjusted to pH8.5. Void volume was determined using blue dextran 2000.The fractions were monitored at 280 nm.

The pigment fractionation was carried out on an IscoModel 328 Fraction Collector, using an ISCO Type 6 OpticalUnit 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 thescan mode (200 through 750 nm). In general, melanin had amuch lower extinction coefficient in the visible range than inthe ultraviolet, making dilutions of the samples necessary foranalysis in the range of 200-350 nm. The absorption spectraof glutaraldehyde-treated cultures were also determined. Using a second basic method of extraction, crystallized pigmentwas obtained by a procedure in which the liquid phase of aculture supernatant was boiled off and the "residue" was driedat 90°C.

Another experiment was designed to determine whether asignificant amount of pigment was cell-associated, orwhether most of the pigment was excreted. Cell pellets(2500 x g, 15 min.) were resuspended in phosphate bufferedsaline (PBS), sonicated at low speed (setting 30) for 30 seconds (Bronwill Biosonik IV Sonicator) and recentrifuged.

10

This pigment preparation was compared spectrophotometri-cally to a culture, containing both cells and soluble pigment,treated in the same way with sonication. Standard solutionsof melanin (Sigma) at a concentration of 0.25 mg/ml distilledH20 and L-DOPA (Sigma) at a concentration of 1.0 mg/mlwere compared with the absorbance spectra of LST culturepigments.

Pigment solubilities were preliminarily tested, using 0.5ml culture supernatant to 2.5 ml solvents. The solutions wereagitated and maintained for at least 30 min after which theywere centrifuged to separate potential precipitates. The criteria of Zussman et al. (1960) were adopted to describe thesolubility 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 notdissolve, and "insoluble" if no color was imparted to the solvent. Solvent-pigment combinations were also examined byspectrophotometer.

Infra red (IR) spectra were determined in collaborationwith 3. Beadle and G. Gokel (Perkin Elmer 281 IR spectrophotometer). Experimental samples were column purified, dialyzed, freeze dried LST pigment from culture supernatent towhich one drop of paraffin oil was added. Commercially obtained melanin (Sigma), synthesized via the photooxidation ofL-DOPA, was used as a control.

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 byMcCardell (1979). Logarithmically growing cultures of LSTwere suspended in 0.066M PBS to an approximate concentration of 2xl09 cells/ml. EMS was added to 1 ml aliquots ofculture to yield final concentrations ranging between 10-30ul/ml (5 ul intervals). The resulting suspensions were incu-

11

bated for 1 and 1.5 hrs in a G76 Water Bath Shaker (NewBrunswick 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 wereinoculated in AG Broth and incubated 2-5 days. After thisadaptation period, the mutagenized and recovered culturewas then spread on Marine Agar. The remaining 1 ml oftreated suspension was used to "spread plate" directly on AGand Marine agars. Screening of mutants was assessed visually, since pigment production was easily scored on agar plates.

ICR 191

The procedures were modified slightly from those described above and by Roth (1975). The reaction mixture consisted of AG minimal medium containing 10% Marine Broth,3-6 x 10" LST/ml and 10 ug ICR 191/ml. Cells were incubated at 30°C in the reaction mixture for 12 hr and then diluted 1:100 into fresh Marine Broth to provide an adaptionperiod of between 12-72 hr. Mutants were screened on Marine, AG and AGT agars.

Shellfish Attachment

In situ studies were done in collaboration with M.A.Emala at the University of Delaware, Mariculture Laboratories. One such study is reported here.

Three spat setting tanks were filled with seawater (25°C)and presetting (eyed) oyster larvae. Acid cleaned (IN HC1,2* hrs) and sterilized glass slides were immersed in Set Tank1. Glass slides, categorized and treated as-follows, wereplaced in Set Tank 2:

1. Pigmented LST: Slides were immersed for 2* hrs ina stationary phase culture of LST, grown in MarineBroth at 25°C.

2. UV irradiated LST: Slides were immersed for 2* hrsin a late stationary phase culture of LST, grown in

12

Marine Broth at 25°C. The slides were then exposed to lethal doses of UV radiation.

3. Marine Broth control: Slides were immersed in un-inoculated media for 2* 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.

*. 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°C) wascentrifuged (2500 x g, 10 min.) and the superna'tantfiltered through 1.2 nm Millipore filters to furtherremove cells. Slides were immersed in the cell freefiltrate for 2* hrs.

After 2* hrs in the setting tanks, all slides and plateswere removed and the attached spat were counted using astereoscope (lOX; Baush and Lomb).

One caveat must be noted. The pigment coated slidesand all of the Agar plates were placed in one tank. TheDOPA 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 populationmay have been enhanced. This suggested that future experiments should be designed to more completely consider DOPAsolubility and autooxidation.

13

RESULTS

Formulation of a Synthetic Medium

LST, though heterotrophic, has relatively simple nutritional requirements (Table 2). Although serine and methionine alone did not support the growth of LST, aspartic andglutamic acids, in combination with serine, methionine oreach other, did sustain the organism. Therefore, we positedthat either aspartic or glutamic acid could serve as a carbonand energy source. In practice, however, medium containingaspartic acid and inorganic salts solution became growthlimiting after numerous subcultures, a situation that was remedied when Asp medium was supplemented with glutamicacid. The resulting AG Synthetic Medium sustained LSTgrowth well and was very useful in subsequent experiments.

LST is a typical marine bacterium in that it utilizesamino acids but not carbohydrates, fails to ferment sucrose,mannose and arabinose (Table 1), and fails to grow in mediumcontaining 1% glucose and salts solution.

As anticipated, LST incurred a lengthy lag period (27 hrs)when transferred from Marine to AG medium. This lag periodwas not observed when cultures were transferred from AG toAG medium (Fig. 2). The generation time of LSTat 25°C was* hrs in marine broth and 7 hrs in AG Medium. The slowergrowth rate in the Synthetic Medium is presumably correlatedto the availability of nutrients in the two media. All growthfactors must have been synthesized de novo from glutamicand aspartic acids in AG, whereas Marine Broth was repletewith numerous vitamins from yeast extract and a wide variety of nutrients in peptone.

Morphology and Morphogenesis

The development of LST was studied under both phasecontrast and scanning electron microscopies, revealing a veryinteresting life cycle. Cells from "young" cultures appearedas very motile, regularly shaped rods, between 1-2 um long

1*

and about 0.2 urn wide. Motile cells had polar flagella whichaggregated in clumps when sheared from the cells. Attachedcells were fixed to surfaces either at one pole or throughoutthe entire cell surface. Attachment fibrils were very prominent. Scanning electron micrographs did not uncover any unusual surface features, such as pili, stalks or other structures.

As the culture aged, the cells elongated, and some became part of chains of dividing yet unseparated individuals.The elongated cells, though they did not divide, formed septa,and many reached well over ten times their original length.As the culture continued to age, the cells became commen-surately less motile, until they became nonmotile altogether.Cells became thinner and more irregularly shaped. Most interestingly, what looked like "bead structures" under phasecontrast appeared within the body of the cells. The beadingprocess usually began at one end and progressed to the otherend of the cell.

Scanning electron micrographs of these "aberrant cells"provided a much clearer understanding of the morphologicalchanges incurred (Figure 1). Indented regions began to formin a slanted orientation along the cell surface. One end ofthe cell often appeared slightly larger in size, suggesting theorigin of the beading process. With time, the indentations became deeper, and the cells eventually took the shape of a regular, tightly wound spiral. No break in the cell wall was observed. Cells displaying this morphology were never motile,and have not multiplied in culture though they maintained anenergy charge (Table 3; M. Emala, personal commmunication)which was characteristic of viable cultures (Knowles 1977).No buds and no empty shells have been seen to indicate acloser similarity to the life cycle of Bdellovibrio, which ismuch smaller in any case. For all practical purposes, thisstage in development appears to be terminal.

Pigment Characterization

After LST cultures reached stationary phase, a solublepigment, ranging in color from reddish-brown to dark brown,became evident. It was retained in dialysis and was precipi-

15

tated by acidified water, ethanol and methanol (Table *). Thepigment was relatively soluble in water, only slightly solublein ethanol and methanol and insoluble in acetone, chloroform,cyclohexane and ethylene dichloride.

The crude pigment exhibited three maximum absorbanceintervals at 260, *07 and the largest at 220 nm (Table 5,Figure 3). Glutaraldehyde partially oxidized the pigment,shifting the absorbance peaks to 233, 273 and *35 nm. Whenthe pigment was totally oxidized, it appeared darkest and anabsorbance peak was shifted still further from 273 to 293 nm.Additionally, there was generalized absorption in the visibleregion.

We compared the absorption spectra of the experimentalLST pigment with spectra of commercial melanin, which hadpeaks at 225 and 273 nm, and with L-DOPA, which had peaksat 233, 282 and 512 nm. The LST product absorbance maximawere deemed to sufficiently match those of the commercialpreparation to conclude that LST did indeed produce a melanin. Further purified LST pigments tended to support thathypothesis. Pigment fractions obtained from Sephadex G-75and G-150 columns yielded absorbance maxima at 226, 263and *07 nm (Figure *). A peak in the visible region was notdetected in the commercial pigment preparation, possibly dueto the consequence of the low solubility of melanin (viz., aparticle-free suspension was not sufficiently concentrated).

Mutagenesis

Ethyl Methane Sulfonate

Mutagenesis with EMS for 1 hr reduced the viability ofLST 2-3 logs as determined by spread plate counts on MarineAgar (Table 6). No colonies formed on AG Agar when LSTwas "plated" directly after mutagenesis. This result was notunexpected: Since the minimal medium lacks so many growthfactors, auxotrophic mutations would be conditionally lethal. Mutagenized suspensions, after 2-5 days "holding" periodsin AG Broth, were streaked on Marine and AG agars. 9Spreadplate counts on Marine Agar ranged from 1.3-6.6 x 10 , while

16

they were approximately two logs lower on AG Agar: l.*-6.5x 10 (Table 6). The colonies on AG Agar were probably inpart progeny of cells that remained in stasis in the AGMedium, repairing damage to the chromosome and possiblyeven back mutating.

Suspensions treated with EMS (all concentrations) for 1.5hrs 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 orminimal media. A number of factors may have led to thisfailure. Two of the possibilities, not mutually exclusive, arethat pigment production is part of an obligate cell survivalpathway, a serious consideration since melanin is part of thetyrosine metabolism. In this case, obtaining pigment-lessvariants may prove an unrealistic goal. A second possibilityis based on reports (Nicolaus 1968) that pigment synthesis isessentially dependent upon a single enzyme, tyrosinase or atyrosinase-like derivative. In this instance, mutations involving the mel gene would appear with very low frequencies.Furthermore, the likelihood of another mutational aberrationthat would be lethal to cells containing a lesion in a mel genewould be high. In any case, we had only screened about 5000colonies by this procedure, and a mutation rate of less than0.02% is not uncommon (Stent and Calendar 1971). Mutagenesis experiments using ICR 191 were designed with a holdingperiod in Marine Broth rather than AG Broth to minimizeauxotrophic lethality.

ICR 191

A total of 2*,803 colonies were screened. Thirty ninecolonies varied in pigmentation, seven had no pigment (hypo),two were darker (hyper), two were light tan, 27 were variousshades of red and one was yellow. The paucity of pigmentmutations 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

17

pigment being a melanin. These results also suggest that LSTmay produce more than one pigment, the lighter ones beingmasked 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 ofauxotrophic mutations were produced. Inexplicably, only 66%of the colonies that grew on Marine Agar, grew on AGT Agar.

Shellfish Attachment

Experiments involving LST or LST product and oysterspat interaction are still quite preliminary. However, datasuch as those reported in Table 7, together with other evidence (M.A. Emala, personal communication), supports thenotion that LST pigment promotes shellfish attachment.Slides coated with pigmented LST attracted more than 5times the oyster spat than the clean and control slides (Table7). Interestingly, slides coated with UV-irradiated LST attracted slightly less spat than the controls, possibly becausethe melanin was photooxidatively degraded (Blois 1965).

The data involving Agar plate imbedded with the testsubstance and glass slides coated with culture pigment are tobe interpreted much more cautiously, since the DOPA diffused out of the Agar, autooxidized, infiltrated the tank and interfered with experimental gradients. Nevertheless, melaninAgar plates also attracted more spat than the control plates,while DOPA Agar plates attracted 2-5 times less spat thanthe controls. The number of spat (attached oysters) was inversely proportional to the concentration of DOPA in theAgar, suggesting that at high concentrations, DOPA may havea 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 controlslides.

18

SUMMARY

These studies of LST reveal a very interesting, potentially ecologically significant organism. The nutritional requirements of the aerobic, salt dependent marine bacterium is satisfied by aspartic and glutamic acids, but not by carbohydrates. The LST life cycle includes normal, dividing, motilestages, as well as an apparently terminal stage characterizedby morphological changes such as spiraling and elongation.The reason for the onset of these changes is not yet known,but they occur in the decline phase of growth and can be induced by toxic compounds swich as benzene, fuel oil, tri-methyl benzene and 2,*-dinitrophenol (A. Segall, unpublisheddata).

In stationary phase, while still physiologically active(Table 3), LST produces a pigment which has spectrophotome-tric solubility and chemical properties similar to melanin pigments described by Irvins and Holmes (1980), Zussman et al.(1960) and Nicolaus (1968). These properties are summarizedin Table 8. It may be that some pigment is produced beforethe stationary phase of growth but in amounts too low to beidentified by spectrographic means. We think, however, thatmelanogenesis is turned on during the later growth stages. Infact, Nicolaus (1968) has reported that small amounts ofDOPA are necessary to prime the first step of melanogenesis,which is the conversion of tyrosine to DOPA. Zussman (I960)attributed the late production of pigment to the lag caused bysynthesis of the catalytic amounts of DOPA by an alternatepathway. Since the extent of melanin polymerization is typically highly variable and is probably a function of the biosyn-thetic system in which the pigments occur, it is to be expected that the LST pigment properties not be identical to other,especially chemically synthesized, melanins.

As a working hypothesis, we consider that melanin production in marine bacteria may be directly related to shellfish attachment. Preliminary studies support the theory thatthe melanin pigment of LST attracts oyster larvae to surfaces. The concentration of pigment excreted by the bacteria and the optimum concentration of melanin, and possiblyL-DOPA, inciting a response in the oysters have not yet been

19

L-DOPA, inciting a response in the oysters have not yet beendetermined. Concentration effects such as these, however,may provide a clue to the exact mechanism of attraction(viz., does L-DOPA act hormonally).

Although we have not yet obtained any stable mel" mutants, their acquisition would be most rewarding in establishing beyond doubt the involvement of the pigment in shellfishattachment and in elucidating the pathway of pigment production in LST. Aside from inherent interest in determiningthe melanin biosynthetic pathway, the information would alsobe of value in identifying the actual compound responsible forthe attraction of oyster spat.

The possibility of a symbiotic relationship between theoyster and LST has not been investigated here, but is of realconsequence. Although bacteria are part of the flora of theadult oyster, they have also been reported to have detrimental effects on the growth and development of oyster embryosand larvae (Walne 1958; Brown 1973). Increasing Vibrio contamination of oysters, resulting in cholera-like diseases inhuman victims, has also been documented (MMWR 1978 27:3*1). A symbiotic relationship between a possible Vibrio andan oyster species may help explain the increased incidence ofdisease.

In conclusion, further research with the LST organism appears highly rewarding from numerous standpoints, includingthe genetic, physiological, clinical and ecological areas of microbiology. This organism promises to be a particularly goodmodel in the study of the consequences of bacterial metabolism on the environment.

20

Table 1.

Some biochemical and physicalcharacteristics of LST

Test Reaction

tGram stain gram neg

I Cell shape rodSporesMotility +Catalase +

Lysine decarboxylase +Ornithine decarboxylaseSucrose fermentation

Mannose fermentation

Arabinose fermentation

Growth in 2.5% NaCl +Growth in 5.0% NaCl +

Growth in 7.5% NaCl +

P

21

Table 2.

Contributions of aspartic acid (asp),glutamic acid (Glu), methionine (Met) and

serine (Ser) to the growth of LSTa

Amino Acids Growth

Asp Glu Met +++Asp Met Ser +++Glu Met Ser +++

Asp Ser +++Ser Met

Asp Glu +++Asp Met +Ser Glu ++

Glu Met ++

Inorganic salts solution (Appendix I) was supplemented witheach of the amino acids listed in the concentrations used inthe AGMS medium.

+++, ^ 7 hr generation time; ++, <^ 10 hr generation time;+, •/* 13 hr generation time; -, no growth.

22

Is)

Table 3.

Adenosyl nucleotide pool in a hypo-pigment producingvarient of LST cultivated in batch culture3

Growth

Phase

LogStationaryStationary-Decline

Viable

Count (cfu/ml) Morphology AECpM

ATP/Cell ug/Cellc

*.5x 107 Short Rods 0.86 1.19 x 10"7 6.56 x 10" u2.9 x 109 Rods 0.80 1.73 x 10'9 9.53 x 10"132.7 x 107 Long Spirals 0.72 1.61 x 10"9 8.86 x 10"13

Cells were removed from a batch culture of LST during log phase, stationary phaseand during the stationary-decline transition and were then frozen (-70°C). Adenosylnucleotides were extracted in boiling tris. The samples were then assayed for ATP,ADP and AMP and adenylate energy charge (AEC) was calculated.

Calculations were based on known internal standards that revealed recovery andcounting efficiencies of 72.*% for ATP, *1.6% for ADP, 38.0% for AMP.

Vg ATP per cell was calculated by multiplying pM/cell by 10"6 and by ATP mol. wt.

Is)

Table *.

Solubility of LST excreted pigment (inspent medium) in seven solvents

Solvent Solubility3 Precipitate"Absorbance0

Maxima

H20, pH 3 S +(2*h) 26*, *01

H20, pH 9 S - 26*, *07

Acetone I +(30s) none

Ethanol SS +(30s) 25*, 375

Methanol SS +(30s) 2*6, 26*, *00

Ethylenedichloride I _ 233, 26*, *00

Chloroform I - 2*3, 276

Cyclohexane I -203, 222

aS-relatively soluble; SS-slightly soluble; I-insoluble

k+ Precipitate formed (time at which formed)- No precipitate formed

cAbsorbance maxima of pigment-solvent mixtures vs.solvent references

Sample

Table 5.

Spectral absorbances of pigmentsextracted from LST

Dilution

Absorbance

Maxima3

Marine Broth

Supernatant1:8

I:*

260

*07

LST-Associated

Pigment1:16

1:4

26*

*07

LST-Associated and

Soluble Pigment1:6*

1:*

260

*07

Red-Black PigmentTreated w/ Glutb

1:6*

1:1000

237

293

Dark Orange Pigment-Glut-

1:6*

1:6*

1:4

23*

272

*36

Yellow Pigment-Glut-

1:6*

1:6*

1:4

232

27*

*3*

Optical densitiesat Abs. Maxima

1.10

0.50

0.36

0.33

0.38

0.25

1.08

0.2*

1.3*

0.58

0.60

1.17

0.38

0.22

to>4

Orange Pigment-Glut-

1:6*

1:2

265

436

Crude PigmentExtract^

1:4 256

none 405

Commercial Melanin0.25 mg/ml

1:4

1:4

225

273

Commercial L-DOPA1.0 mg/ml

1:4

1:4

none

233

282

512

2.00

0.34

1.16

0.11

0.43

0.31

3.00

2.900.37

aThere were 2-3 maxima for each sample. See text and Figure 3 legendfor further detail.

bGluteraldehyde, an SEM fixative.cCrude pigment extract was obtained by redissolving crude pigment crys

tals in distilled water to solubility limit (exact concentration unknown).

Is)00

Table 6.

Toxicity of Ethyl Methane Sulfonate(EMS) to LSTa

EMS Cone. Direct Growthb Growth after Holding0ug/ml MA AG MA AG

10 3.7xl06 No Datad 1.4xl09 1.5xI0715 1.3x10s ii No Data No Data20 7.0xl07 n 6.6xl09 6.5xl0725 6.9xl07 ii 3.2x10s 2.1xl0730 7.1xl07 ii 1.3xl09 1.4xl07

originalculture 6.7xl09 3.8xl09 ~ "

aLST was exposed to EMS concentrations for 1 hr.Mutagenized suspensions were spread on plates immediately after exposure toEMS.

cAliquots of mutagenized suspensions were "held" in AG Broth for 2-5 days, afterwhich they were spread on Marine (MA) and AG agarsThe dilutions plated did not yield any colonies.

Table 7.

Density of Crassostrea virginica larvaeattached to glass and agar surfaces

Slide or Agar Plate Attached SpatPreparation3 Density

Clean and Marine Broth 0.11/in 2 (16)Control Slides (I)

Pigmented LST (II) 0.58/in2 (16)UV Irradiated LST (II) 0.07/in2 (16)

Culture Pigment (III) 1.00/in2 (10)10 mg DOPA/ 10 ml agar (III) 1.03/in2 ( 2)50 mg DOPA/ 10 ml agar (III) 0.42/in2 ( 2)

100 mg DOPA/ 10 ml agar (III) 0.*2/in2 ( 2)

10 mg melanin/ 10 ml agar (III) 2.22/in2 ( 2)20 mg melanin/ 10 ml agar (III) 2.55/in2 ( 2)Agar Control/ 10 ml agar (III) 2.12/in2( 2)

aNumber in parentheses designates the spat tank used.

Number in parentheses designates the number of samplestaken.

29

Table 8.

LST properties of pigment compared with pigmentsidentified as melanin of other microorganisms

PROPERTIES ORGANISMS

Aeromonas3 Vibriob Aspergillus0 LSTdliquefaciens cholerae nidulans

Color Brown-Black Brown Black Brown

Solubility in H,0 at pH 7Solubility in 0.1 N NaOH

I ND ND SS

S S S S

Blackberg-Wanger Precipitation PPT PPT ND PPT

FeCl-Precipitation PPT PPT PPT PPT

Acid Precipitation PPT PPT PPT PPT

v*> Reduction (Glutathione) + + + +

oReoxidation + ND + +

Absorption Peaks Diffuse 345,480 480,535 264,407H20 Bleaching ND + + +

Molecular Weight ND ND 2,000,000350,000

29,000

120,00052,00012,000

I - insoluble; S - soluble; ND - no data; PPT - precipitated; +

3 Aurstad and Dahle 1972b Wins and Holmes 19800 Bull 1970

Present study

positive

a. Normal rod structure.

b. Forming of slanted indentations across the cell surface.

Figure 1. Scanning Electron Micrographs of the Morphological Changes of LST.

31

c. Advanced stage in spiral formation.

d. An elongated cell with spiral forms in the background.

Figure 1 continued.

32

e. General view of aberrant vs. normal cell morphologies.

Figure 1 continued.

33

200

O

10

0

20

02

50

TIM

E(H

OU

RS)

Figu

re2.

Gro

wth

Cur

ves

ofLS

TC

ultu

res

Gro

wn

inM

arin

ean

dA

GSy

nthe

tic

Bro

ths.

Cul

ture

sof

LST

wer

egr

own

inM

arin

eB

roth

and

AG

Synt

heti

cB

roth

for

470

hrs,

duri

ngw

hich

tim

eth

eyw

ere

peri

odic

ally

mon

itor

edus

ing

aK

lett

-Sum

mer

son

Col

orim

eter

.Lo

g^0

O.D

.w

asp

lott

edve

rsu

sth

eti

me

atw

hich

the

read

ing

was

tak

en.

•MARINE

BROTH

CULTURE

•PRIMARY

INOCULATION

FROM

MARINE

BROTH

TO

AG

BROTH

•INOCULATION

FROM

AG

BROTHTO

AG

BROTH

9 \

2.0

1.8

1.6

1.4

A / \•«>i < •• i\ < i• i\ i i• »\ * t• 'i • .

'\V \

CULTURE PIGMENT

PIGMENT TREATEDWITH GLUTARALDEHYDE

1 .2 • \ *• \ *• \ %

ca1.0

o • \ t• \ •• \ *« \ ^_ - _^

.8 » ^->^^ X» Xt \1 \

.6 N \ \\ * \\ 1 >

.4\ 1

Xv %

.2

n

%% *

'

UV 200 220 240 260 280 300 320 340 360

VISIBLE 350 400 450 500 550 600 650 700 750

WAVELENGTH IN NM

Figure 3. The Absorbance Spectra of a Culture Pigment anda Glutaraldehyde-Treated Pigment. LST grown inMarine Broth to stationary phase was centrifugedat 10,000 xg to "pellet" the cells and thesupernatant was analyzed using a Beckman Model25 Scanning Spectrophotometer. The glutaral-dehyde treated pigment was obtained from aculture originally prepared for SEM. The culturesupernatant was again analyzed using the spectrophotometer in the scan mode. Dilutions weremade when necessary to obtain a peak between0.0 and 2.0 optical density. (NOTE: Use UV scalefor upper curves, visible scale for lower.)

35

OS

a

o

1.0 Y

.8 •

.4 •

UV

VISIBLE

WAVELENGTH IN NM

Figure 4. Absorbance Spectrum of a Pigment Fraction Purified on a Sephadex G-75 Column.Supernatants from stationary phase cultures of LST were dialyzed overnight againstdistilled water and passed through a Sephadex G-75 column. The pigment fractionswere then analyzed using a Beckman Model 25 Spectrophotometer in the scanmode. (NOTE: Use UV scale for upper curve, visible scale for lower curve.)

100

CO

LST PIGMENT

'MELANIN^

co 1)0 •

ce:

4000 3500 3000 2500 2000 1800 1600 IdOO 1200 1000 800 600

WAVENUMBER (CM"1)

Figure 5. IR Spectrum Pattern of Melanin and LST Pigment.

37

LITERATURE CITED

Adler, R.J. 1966. Science, 153: 708.

Aurstad, K. and H.K. Dale. 1972. The production and someproperties of the brown pigment of Aeromonas liquefaci-ens. Acta. Vet. Scand. 13: 251-259.

Barnes, J.R. and J.J. Conor. 1974. The larval settling response of the lined chiton Tonice11a lineata. Chemore-cept. Abstract 2: 25.

Belas, R.M. and R.R. Colwell. 1982. Adsorption kinetics oflaterally and polarly flagellated vibrio. 3. Bacterial 151:1568-1580.

Blois, M.S., A.B. Zahlan and J.E. Maling. 1964. Biopys. 3.,471.

Brown, C. 1973. The effects of some selected bacteria onembryos and larvae of the American oyster, Crassostreavirginica. 3. Invert. Pathol. 21: 215-223.

Bull, A.T. 1970. Chemical compositions of wild type and mutant Aspergillus nidulans cell walls. The nature of polysaccharide and melanin constituents. J. Gen. Microbiol.63: 75-94.

Colwell, R.R., J.B. Kaper and S.W. Joseph. 1977. Vibriocholerae, Vibrio parahaemolyticus and other Vibrios: Occurrence and distribution in Chesapeake Bay. Science198: 394-396.

Corpe, W.A. 1970. Attachment of marine bacteria to solidsurfaces. In R.S. Manley (ed.), Adhesion in BiologicalSystems. Academic Press, New York, p. 73.

Crisp, D.J. and J.S. Ryland. 1960. Influence of filming andof surface texture on the settlement of marine organisms. Nature 185: 119.

39

Havenner, J.A. 1976. A defined medium fulfilling the growthrequirements of Hyphomicrobium neptunium. M.S.Thesis.

Havenner, J.A., B.A. McCardell, and R.M. Weiner. 1979. Development of defined, minimal, and complete media forthe growth of Hyphomicrobium neptunium. Appl. Environ. Microbiol. 38: 18-23.

Ivins, B.E. and R.K. Holmes. 1980. Isolation and characterization of melanin-producing (mel) mutants of Vibrio cho-lerae. Infect. Immun. 27: 721-729.

Kirby, G.W. and L. Ogunkoya. 1965. Chem. Commun. 21:546.

Knowles, C.J. 1977. Microbial metabolic regulation byadenine nucleotide pools. Symp. Soc. Gen. Microbiol. 27:241-283.

Larman, V.N. and P.A. Gabbott. 1975. Settlement of Cypridlarvae of Balanus balanoides and Elminius modestus induced by extracts of adult barnacles and other marineanimals. Chemorecept. Abstr. 3: 38.

Levandowsky, M. and D.C.R. Hauser. 1978. Chemosensoryresponses of swimming algae and protozoa. In International Review of Cytology 53: 145-210.

Mason, H.S. 1953. In M. Gordon (ed.) Pigment Cell Growth.Academic Press, New York.

Mayfield, C.K. and W.E. Innis. 1977. A rapid, simple methodfor staining bacterial flagella. Can. J. Microbiol. 23:1311-1313.

McCardell, B.S. 1979. An approach for the recovery of thymine requiring mutants of Hyphomicrobium neptunium.Ph.D. Thesis.

40

Meadows, P.S. and G.B. Williams. 1963. Settlement of Spi-rorbis borealis Daudin larvae on surfaces bearing films ofmicroorganisms. Nature 198: 610-611.

Miiller, W.E.G., R. Zahn, B. Kurelec, C. Lucu, I. Muller and G.Uhlenbruck. 1981. Lectin, a possible basis for symbiosisbetween bacteria and sponges. J. Bacteriol. 145: 548-558.

Nicolaus, R.A. and M. Piatelli. 1965. Rend. Ace. Sci. Fis.Mat. Napoii, XXXII, 82.

Nicolaus, R.A. 1968. Melanins. Hermann, Paris.

Robson, N.C. and G.A. Swan. 1966. Symposium on structureand control of the melanocyte, Springer-Verlag, Berlin.155.

Roth, J.R. 1975. Genetic techniques in studies of bacterialmetabolism. Methods of Embryology 174: 3-35.

Stent, G.S. and R. Calendar. 1971. Molecular Genetics.W.H. Freeman and Co., San Francisco.

Swan, G.A. 1964. Rend. Ace. Sci. Fis. Mat. Napoii, XXXI.

Walne, P.R. 1958. The importance of bacteria in laboratoryexperiments in rearing the larvae Ostrea edulis (L.) J.Mar. Biol. Assoc. U.K. 37: 415-425.

Zobell, C.E. 1943. The effect of solid surfaces upon bacteriaactivity. J. Bacteriol. 46: 39-56.

Zussman, R.A. I. Lyon and E.E. Vicher. 1960. Melanoid pigment production in a strain of Trychophyton rubrum. J.Bacteriol. 80: 708-713.

41

APPENDIX I

COMPOSITION OF THE AGMS AND AGSYNTHETIC MEDIA

The AGMS Synthetic Medium (Havenner, et al. 1976)consists of two stock solutions:

Stock //I:

NaCl 19.45 g/LMg.CI,.6H20 8.80 "Na.SO. 3.14CaCL (anhydrous) 1.80 "KC1 0.55NaHCO, 0.16KBr . . 0.08H-BO. 0.022

SrCL 0.034NaSiOj 0.004NHuNO, 0.0016 "Na^HPO,, 0.008Ferric Ammonium Citrate 0.10 "

The salts solution is autoclaved at 121°C for 15 min. at 15 lbspressure. Sterile solution should be stirred to evenly distribute the precipitate formed.

Stock //2:

Aspartic Acid 26.27 g/LGlutamic Acid 23.77 "Methionine 0.39 "

Serine 17.16

43

The pH of the Stock //2 solution needs to be adjusted to 7.2-7.4 using 6N NaOH. Sterilization by autoclaving as above follows pH adjustment.

AGMS Medium consists of a mixture of 30 ml Stock tilwith 70 ml Stock #2.

AG Medium uses the amino acid pool given below:

Stock //3:

Aspartic Acid 26.27 g/LGlutamic Acid . . .. 23.77 g/L

Stock til and Stock #3 are mixed in the same proportions asfor AGMS Medium (30-70) after adjusting the pH of the solution to 7.6 with 6N NAOH and sterilizing the solution.

Stock //4: Phospate Solution

K2HPO„ 13,6 g/LK2HPO„ 21.3 g/L

Autoclave separately, add 0.46 ml/100 ml GAMS

44