FROM UNC-CH CON' RACTS 9 GRANTS 09.13.1994 15142 P

P~a ~AT~~I ~( #cr.n Approvifd

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1. AGEN-CY-V U. 'ONLY rLeelv. bldnk) 12. PORT DATE OI REPOAT TYPE AND OATES COVERED________________ 9/20/94 Fnl(Z.-15/93-4/14/94)

- 4. 711LE AND aETITILt -K 5, unr6N umsekG "I-,

NIMolecul r Interactions at Marine Interfaces N00014-0-3-1- ',IC~

00 .AUNFS

S (JRalph S. QuatranoW,7. PFRFORMIN 1I~R6ANIZATION NAPAF(9) AND ADDRIESS(ES) B 7PrFrORMJING ORIGANIZATION

Univ. cf North Carolida at Obiapel Hill REPORT NUMBER

Departfent of Biology!CB #32W -Coker Hall1Chapel Hill, NC 2759)-'3280

All

g. SPONSORIN /OI0NG AGtNKY NAMEWS AND ADDRISS(ES) 94-31571Contracts and Grants :11111iill i'tl'ii l440 W. Franklin Street htii lIi HIl l'I~IChapel Hill, NC 27599-1350

11. SUPPLEME 1ARY NOTES

Workshol schedule, abstracts and li-:t, of partici pants attachied.

12s. DISYRIRUI ISN IFAVAILABILITY STMtMMENY 12b. DISTRIBUTIONJ CODE

Unlimi' ed Un l imi ted

13, ABSTRACT (MaximurnI200words)

Approximately 25 scientists from around the world participated\- in a workshopaimed at dfiscussing) molecular interactions at mar-ine interfaces fromI two

pe~rspect'yes: the cheicnistry of adhesive polynier., anid the b~iology of structutcsthat ser-e as attachmnent organs; of marine orgam-sns. Detailed presentations ofeach area was complcxncnted by joinit discussions of the. two perspectives hin orderto achie -e interaction on the research pro~~gras supported by ONR.

14. S~tt f ms15. NiUMBER OF PAGES

Adhosi -! x, hi olbul im, adhesive polym~ers -Aý16. P1Rict COWII17. SECURITY (IAiSIICkTION 18,.:SECURITY CLASSIFICATION 19. SECUR17Y CLASSIFICATION 20T, LMIIATION OfAUSIRACT

OF ~ ~ O THI Io 14$ PAGE OF~ APSIRACT

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THLE UNIVERSITY OF NORTH CAROLINAAF

CHAPEL HILL

Depar'mens of Biology CBJI 3280, Coker fiat]College of Arts and Sciences The University of North Ca.-olita at Chapce Hill(919) 962-3775 Chapel -Hill, NC 2;599-3280FAX - (919) c,62-1625

September 26, 1994

To Whom It May Concern:

SUBJECT: N00014-93-1-0491

Enclosed is my report of a workshop held at The University of North Carolinaon April 20-22, 1993, entitled "Molecular Interactions at Marine Interfaces".Included is a Report Documentation Page (Form 298) and a copy of the WorkshopProceedings.

If I can be of any funtler assistance, do not hesitate to contace me.

Sincerely yours,

Ra S. Quatrano

John N. Couch Professor and ChairAccesion For

NTIS CRIA&I #D1IC 1IA13 %cc: Mr. Scott Blackwood U- 1,,ou iccd Li

Contra( s and Grants J: ., 1on.UNC-CH

By ... .... ..................

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Grant No.: N00014-93-1--0491

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NPIAVAL •

Molecular Interaotions at Marime InterfacesFriday Center, University of North Carolina

April 20-22, 1993

V1

Molea-ar- itera ons at Marine Interfaces (MIMI)Friday Center, University of North Carolina

April 20-22, 1993

Tuesday April 20th8:30 Hal Guard & Randall Alberte: Welcome and Program Overview

Mivtimally Adhesive Polymers9:00 Ken Wynne Minimally Adhesive Polymers - Overview

9:30 Joseph Gardella Surface Chemistry of Multicomp.nent Polymers forPotential Non-Fouling Coatings: Air and MetalInterfacial Issues

10:00 Toby Chapman Segmented Polyurethanes

10:30 Coffee Break

11:00 Chris Ober Self-HIealing, Low Energy Polymer Surfaces using BlockCopolymers

11:30 Aslam Malik Future Direction in Oxatane-derived Polymers

12:00 Lunch

1:00 Jim Griffith Synthesis and Evaluation of Fluoroepoxy Resins Curedwith Amine-terminated Polydimethylsiloxane Oligomersfor Use in Marine Environmenis

1:30 Kurt Baum Pclyuethanes Based on Hybrid FluorocarbonPolysiloxanes

Floadhesive Surface htteraiois2:00 John Rabolt Characterization of Polymer Surface Interactons by

Spectroscopic Techniques2:30 Tom McCarthy Chemical Control of Adsorption at Polymer-Aqueous

Solution Interfaces3:00 Coffee Break

3-30 Georges Belfort Direct Measurements of the Intermo'ecular Forcesbetween Polysaccha ride Exopolymers from MarineBacteria and solid Substrates

4:00 Dave Hartsough Development of Theoretical Tools for StudyingCarbohydrate Structure, Dynamics and Sugar-SurfaceIn teractions

4:30 Open Discussion

5:30 Wine & Cheese

Wednesday April 21st8:30 Dave White Effect of PolysacchaJ-.-e Surface Structure on Microbial

Attachment and Biofilm Formation9:00 Michael Sinnott Structure of Extracellular Polysaccharides of

Pseudomonas atlantica9:30 Herb Waite Interfacial Culprits: Targeting Proteins of Byssal

Adhesion10:00 Ralph Quatrano Presence and Role of a Vitronectin-Like Protein in

Adhesion of Fucus Embryos to Marine Substrates10:30 Coffee Br'eak

11:00 Kyle Hoagland & Biochemistry of Fouling ,'4arine Diatom Adhesives andMichael Gretz the Effects of Substrate Preconditioning on Adhesion

11:30 GiU Geesey Characterization of Molecular Interactions between"Conditioned" Po!ymer Surfaces and Microbial BiofilmMatrix Polysaccharides12:00 Lunch

1:00 David Kirchman Molecular Char'acterization of Specific Attachment by aMarine Bacterium

1:30 Arnanda Goodman Genetic Responses of Bacteria in Biofilms

2.00 Steve Snyder Navy Biofouling Programs

2.:3 Co"nffee BRea,,L-

3:00 Working Session: Emerging Research Directions for MIMI Program

5:30 Wine & Cheese

Thursday April 22nd

9.00 Working Session: Research Collaborations & New Approaches

10:30 Coffee Break

11:00 Plan for next investigators' meeting

11:30 Adjourn (lunch on your own)

2i

Surface Chemistry of Multicomponent Polymers for Pu.AentiaINon-Fouling Coatings: Air and M. etal Interfacial Wirses

Joseph A. Gardella

Studies under-way in our laboratory funded by ONIR MIMI include thie quantitativesurface characterization of various polymers at the air and metal interface. Studies include thesynthesis of surface mcidifications meant to promote and prevent adhesion where appropriate for

L marine coating applications. Analysis utilizes various electron arid optical spectroscopic,mnicroscopic, mass spectroretrv and contact angle measurements of the surface and interfacialcomposition, where appropriate.

Two projects wvill be desci-Ibed in this presentation. Studies of polyureLha- -siloxanesystemns1 (including segmented copolymers and homopolymer-copolymer blends) have beenpursued, with the general goal of prov~iding a quantitative surface and in depth analysis of theamount of surface excess of si loxane chain as a function of segment length, bulk composition

L and proccssing t-eatmtnts (i.e. solvents casting. annealing, etc.). A critical feature of these resultsis the understanding of the factors which govern the surfa,-e excess of siloxane, and these studiescomplement a variety of other systems examined in this laboratoi 2 aloIn cocuIon bu

A-- the nature of interactions between components in a multicomponent polymecr which control theresulting surface excess of a lower energy component. Quantitative surface analysis of thesesystems will be presented.

The s'!cond project presented will focus on the surface modification of fluoropolymers tonrcimnte and rcnnrn-i metal arhe.ion In combination with researchers at the Naval ResearchLaboratory, wt have combined electxoless meta deposition from covalently bound pir-cursorswi'th the controlled lithographic modification of FE:P teflon 3,alwna ipetrespmeldeposition with strong adhesion4,. This process requires no high cost treatmients. consisting oflow wattage plasma treatments and aqueous based- metallization chemistry. At a fundamentallevel. this work shows the value of covalently hound functional group pcecursors to promotingstrong metal adhesion at the interface of a low surface energy polynx~r.

Finally, fuLure wor[- in the development of new sur-face analytical methods. NXtll bcd-scribed. One major goal if this ONR spotnsored programi is to make a,.aikibic thesetechnologies to all MIMI n: ýcarchers.

1. T. Ho, X. Chen. J. A. GardLdta. Jr. arid K. 1. Wynne. ww:k in prugrc-'sX. . Clicn. Hl. F. L-c arid J. A. Gardclla, Jr. "The Effect% o w &L~WC rbnd Ane Ln)ntcSr.

Comnposition of Mult-i-block Copiolyiners of Bisphenol A Polycarhma~te and PONy (DivicthNl Siloxanct.-Maicromokuic~s, submittod [X-c 199?.

3. US Pa--nt #4.946.903-N~codificd Fljorminatzd Polymer ('oipos)itionis Having lncrca-rd Suniac EnergyPropetis" T. G. Vargo and J. A. (hr..dlta. Jr.. Co- ln%-ciiu~rs. D). J. Hlook. 1. G. Vargo. 1. A. (Gank-1la. Jr, K. S.

A Litwiier and F. V. Bright -Silaniz~suon of Radio Frequency (ilmh Discharge Siddiliod Expanided Pul)('ctz-dUraf dii ylx- flC) (OTTE-) Using .artinopropylt; -hox y Siaine (APTL-S)." Lwigrnur. 191.2Z. )42-51- T 6IVargo. P. Ni. Thomnp:on. L. J. CLrcnscr. R. F. Valic.-ini. 1P. Aetnshcr. D. J. Ilotk arid,. A Gardclla. Jr. -NMunola'~erChemical Lithography arid Characteruatonc of Fluoropiovynicr Faihn%.- Langnih ix. 194~2. h. 1 30O 1134.

4. 1'. G. Vargo, 3. A. Gardella, Jr.. anld 1. MI. Cai ert..L Lkxtilcm. 1)cpositiul (if Nlcwkl onto l1uiortoucl~iricSubstrates." Journal of the Ainricn'an Clacrmcal Society. In iweparatao.

Segmented PolyurethanesToby M. Chapinan

Segmnented polyurethanes have been prepared with special soft blocks consisting of N-Akylated polyamaides and polyurethanes. If dhe alkyl substituents ame fluorinated, there. is aprofound effect on hthe surface contact angle with water. Depending upon the method ofmeasurement, contact angles as high as 105-111' have been measuredJ. We. are also preparingamphiphilic dendrite polymers with fluorinated exteriors which we hope wili position themselvesand anchor at the aic interface of a polyether polyurethane.

Self- Healing, Low Energy Polymer Surface Using Block CopolymersC. K. O')er and E. J. Kramer

Self-healing low energy polymer surfaces are being produced by doping higher surface. 01 . energy homopolymers with block copolymers containing one block %%t.ich is miscible with the

homopolymer and another which contains either siloxane or fluorinated moieties. Model iblockcopolymiers have been synthesized by first producing a block copolymer of polystyrene and 1,2-isoprene by anionic polymerization. I lydrosilyaJon ha-s been used to couple siloxanes andappropriately modifie-d fluorinated alkanes to the vinyl groups on the block copolymer. Thesurface ex:cess and segment density of the block copolymer as it segregates to the surface ofmixtures of the block copolymer and 'polystyrene is being measured by forward recoilsnectronicurv (FRES) and RBS. Measurements ef the surface adhesion energy (JKR technique) i.;being correlated with measurtments of the contact angle of water on the surfacc to establish

T. segregation behavior to, and energy of, the resulting polymer surfaces.

Future Direction in Oxatane-derived PolymersAslam M41ik

No abstract available.

Syaithesis and Evaluation of Fluoroepoxy Resins Cured with Arnsi>: -terminatedPolydimethy~isiloxane Oligoiuers for Use in NMarine Environmnents

Ann E. Mera and James R. Criffith

Various difunctional and tr-ifunctional fluoroepoxy re -ins have been cured with twoamine-termiinated polvdlirnethylsiloxane A.lgorners of different molecular weights. The curedfilms have been eharactenzed to determine glass transition temperatures. wa'er contact anglcs,equilibrium water absorptions, and surface adhesivity. For each property the effects of therla'ivc amounts of silicon and fluorine ',n the cured resin are discussed along with compatibilityand tractio beairo h trigmtrials. I hc cured resins are being, evaluated for theirsuitamility as products with exceptional merit for use in marine environments.

4

11olyurethanes Based on Hybrid Fluo;-ozarlvn PolysiloxanesKurt Baum

The Objective of this program i, to prepare and characteriz,- polyurethanes based on diolswith hybrid fluorocarbon -poly s Iloxane backbones. a,w-Diiodoperfluoroalkanes are rea':ted withethylene, and the insertion products are treated with base to give a,w-diolefins, CH2 --CH-Rl1CH=CH2. H-ydrosilyla tion with chlorodimethylsilane gives the chi orosilanes, ClSi(CI- 3)2CH-CH-R I-CH-SHSi(CH3 )2C1. Hydrolys- a gives the correspo~nding silanols, HOSi(CH 3)2C] f2-CH-Rj-CH-CHSi(CH3)20H. Polysiloxar-'- %i-rmation in the presence of 3-trifluoroacetoxvpropyichilorodimethylsilane as an enid--cap gives CF3CO2-(CH2 )Y3 Si(CH3 h [O)-Si(C~I3 )}2CH2ClI 2.-R1 -

CH2GH42Si(CH3)2 -14 0O(CHi2>j-O 2CC'F3. Ester hydrolysis gives the coresponding diol. HO-(CH2}3-Si(C-H3)2 [0-S i(CI13)2CH2CH 2-R1 -CH 2CII2Si(C-i- 3 >2-]4 0(CH12)3-OH. The1ý use of acetaterather than trifluoroaceta-te end groups requirts hydrolysis condition severe enough to reorganizesiloxa~ne units. Elastomeric poly urethanes were obzained with hexamnetlyllene diisocyanaie. withglass transition temperatures of -50'C.

Characterization of P'olym~er Surfacx. Interactions by S pect roscopic TechniquiýýJohn Rabolt

Polarized MTIR spectroso-opy has proven to be an extremely useful tecbhnique for thestudy of molecular topography on surfaces. TRe sensitivity of thic- technique has provided the'Ahily to study mnoiecuks and polymers in films whose5 thickness is on ine )rder (H 1 .- 5-3- nIi.The info-mation obtained can be used to assess the rxcrtit of orie ntation and order in thin filmsand how this changes \khcn a surf ace i!- placed -in aggre. -live environmients. Some of theopportunlties whic:h this ,cchnique provid:cs for the s, udy of proteirmlsurface interactions %kill beýIighlligh.ed.

Chemical Control of Ad~orption at P1olymer-Aqlueous Solutiofi InterfacesThomas J. NfcCar-thy

This tWk will describe chemical strategies to study and control thc. prtxcss of adsortinonof polymers (in particular. po~lysaccharoidcs and model proteins) from aqueous; solution to thesolution - organic polymer interface. 0..ir research involves tothi (1) controlling the snrilkture ofthe solid polymecr surface through the introduction anid fuither chcmistry of vers-atile organicfunctional groups (ichmically r-sistant poxlyniers are used a% ý_Lihstratc.) at the surface and (2)controlling the sLinand itixrface behavior of tiic absorbing polymer fthou.gh chemicalmodification and solution property contic!- Thei tak w16ill describ, our particular areas ofexpertise and the physical -organ ic 1001s that we can use to control interaction% Mt mar-neinterfaces and pinpoint the areas in which collabhorative assistance could benefil our efforts.Specific cx~mplcs of surfacc modification and polymwer ad-sorptiori wxill be discussed ;.ud ourstrategics to control rpolysaccharide adsorption will be outlined.

(4 sit/ i

u~ .

Direct Measurements of the Intermolecular F~rccs Between PolysaccharideExopolymers from marine Bacter"a and Solid Substrates

Georges Belfort

The kinetics and equilibrium ed!orption of an exopolysaccharide (CB115A RsaA EPS),extracted from the fresh water bacteria Caulobacter crescentus, was measured for several modelhydrophobic and hydrophilic surfaces. Changes in advancing contact angle resulting fromadsorption onto these swfaces were determined. Using the surface force apparatus, we plan tomeasutre the intermolecular forces (force-distance profiles) anu concentration-distance profiles(from refractive index) between model EPS from marine and fresh water bacteria and varioussolid substrates such as inorganic rTuca and polymer films with different surface chemistriesunder simulated marine conditions. Various theories will be used to predict the concentration-distance profiles and tested against these measurements.

Development of Theoretical Tools for Studying CarbohydrateStructure, Dykamics and Sugar-Surface Interactions

Kenneth M. Merz Jr. and David S. Har:sough

Our long term goal is to develop and exploit theoretical techniques to under:-tandcarbohydrate dynaa;zs, structure and functioni, and the interaction of saccharides with surfaces.Currently there are no potertial functions available that model intramolecular and intermolecularcarbohydrate interactions for any generalized sequence Furthermore, structural informationconcerning surface -ca-bohý,urate interactions is lacking and therefore information that we canobtain vsing cnmputer simniatinn terhniques will he of great wiu in unnrinr-,tndirngc theseinteractions at the molecular level. In an effort to increase our understanding of these systems,we propose the development of a general molecular mechanical potential function whichaccurately describes intramolecular and intermolecular interactions of carbohydrates. Such aforce field will enable us to model any arbitrary polysaccha-ide in a solution environment and toinvestigate in detail its dynamic properties over time. We also propose using the newly develop-ed force field to study the interactions and forces between individual saccharide monomers andmodel surfaces in an aqueous medium of high ionic strength.

Effect of Polysaccharide Surface Structurt on Microbial Attachment and Biofilm FormationDavid C. White and A. Sonesson

We utilize a flow-through system in which the colonization by bacteria of flat coupons incontrolled laminar flow can be monitored non-destructively by measurement of fluorescence oftryptophane in the proteins. Activity of the Vibrio han'eyi or genetically engineered P._eudo-monas in which the activity of specific operons can be monitored by their bioluminescence.Preliminary tests indicated coating coupons with uronic acid polysaccharides increased adhesionand biofilm fonation over stainless steel. Changes in 'lie chemistry of the polymer-microbeinteractions will be monitored with the attenuated total reflectance/Fourier transforming infraredspxctrometer as the film forms on germanium crystals. Polymers from genetically manipulatedbacteria induce differential bit- qlm formation. Pseudorono.s with the bioluminesccnt lux genecassette %, ith the promoter for algD allow testi ig role of uronic acid polymers in adhesion andgive indications of gene action in the biofilm.

6

Structure of Extracellular Polysaccharides of Pseudvrzonas Atla ticaMichael L. Sinnott

The tide organism is known to change from mucoid to rnon-mucoid forms a's aconsequence of a genome rearrangement. We are investigating the structures of the polysac-charides produced by the mucoid fon-n of the organism. It produces both a neutral and an acidicpolysaccharide. Carbohydrate analysis, and various NMR techniques (DQF-COSY, 13C-1Hheteronuclear COSY, NOESY, polarization transfer) have revealed that the acidic polysaccharidehas a heptasaccharide repeat of five rhamnose units, a b-ribofiuran ose unit and b-glucopyranoseunit, with the acidity probably due to pyravate groups. A rhamnotriose unit is labile, and itscleavage from the main chain results in precipitation of polysaccharide. A provisional structurewill be presented.

Work envisaged for thet immediate future involves firming up this structure, anddetermining the structure of the neutral polysaccharide. In the medium term, questions to beaddressed involve the solution conformations of the hydrophobic anionic polysacchauide and thehydrophilic neutral polysaccharide, with reference to their adhesion properties, and themechanisms of biosynthesis of both polymers.

Interfacial Culprits: Targeting Proteins of Byssal AdhesionHerbert Waite

The adhesive holdfasts of marine macrofouling organisms contain a consortium ofmolecules that are endowed collectively with the enviable property of attaching the animals to awide variety of surface types. To date, nothing but speculation has been advanced to account forthe unusual stability and veisatility of marine holdfasts. Even in mussel byssus, the mostscrutinized of holdfasts, all models regarding the distribution of the 5 major byssal proteins in thevicinity of the interiace remain largely speculative. Although some of the proteins are in fastextraordinarily sticky, there is no direct evidence implicating any of these at the interfa;e.Mussels oblige the study of the plaque/substratum interface by voluntarily depositing byssaladhesive plaques onto any available substrata. This ingenuous opportunism can be exploited byoffering mussels dialysis membranes or other materials with macromolecular porosity. Sincesuch membranes are Dermeable to discrete ranges of molecular dimensions, the molecules at theinterface can be prefelrntially targeted by reacting fouled emersed membranes on their nonfoutedsides with various chemicals used for modifying proteins. Modification should be limited only tothose proteins or parts of proteins accessible by diffusion from the unfouled side. Targetedpeptides will be recovered from partial hydrolysates by conventional IIPLC purification. Peptidesequences will be used to generate specific antibodies to isolate soluble precursors.

7

Presence and Role of a Vitronectin-Like Protein inAdhesion of Fucus Embryos to Marine Substrates

Ralph S. Quatrano

Zygotes of the brown alga Fucus provide a model system study the basic mechanismsinvolved in the generation of zygote asymmetry and directional transport of unique cytoplasmiccomponents to the resulting daughter cells. Since the rhizoid cell of the two-celled embryoattaches the developing embryo to the substratum, this system can also be used to study theprocess of adhesion of this alga to marine substrates.

The rhizoid cell is highly polar and directionally transports (via Golgi vesicles)macromolecules that are secreted into the elongating cell wall or extracellular matrix (ECM).Previous results indicate that a highly sulfated fucan glycoprotein (F2) is locally deposited in theECM of this elongating tip. If F2 is prevented from being enzymatically su!fated (met-embryos),normal two-celled embryos are formed by F2 is not localized in the rhizoid tip and the embryosdo not adhere. Recent results have identified a vitronectin-like (Vnl) molecule with the samepattern of distribution as F2 in the Fucus two-celled embryo. Polyclonal antibodies to humanvitronectin recognize a glycoprotein in extracts of zygotes and two-celled embryos of Fucus witha molecular weight (62-65 kDa) similar to human vitronectin (Vn). The specificity of theimmuno-cross-reactivity is further demonstrated using controls of monospecific and. non-immunec antibodies, and competition by purified Vn. The Fucus Vnl molecule can also be isolat-ed by procedures established for mammalian Vn; glass bead and heparin affinity chromato-graphy. Immunolocalization and subcellular fractionation results demonstrate that Vnl is firstlocalized in the cytoplasm of the zygote and later in the ECM of the elongating rhizoid tip. Met-embryos do not exhibit this localized ECM distribution of Vnl and do not adhere to the substrat-um (similar to the results with F2). In a functional assay, adhesion of Fucus embryos ispreverned in the presence of the Vn antibody, suggesting that the Vnl plays a role in adhesion asin animal systems. Current work is focused on isolating gene sequences that code for the Vnlprotein fromn marine algae as a first step in understanding the molecular basis of adhesion.

Biochemistry of Fouling MWrine Diatom Adhesives and theEffects of Substrate Preconditioning on Adhesion

Kyle Hoagland and Michael R. Gretz

As a prelude to investigations of the physiology and molecular mecbanisrns ofbiosynthesis of extracellular polymeric substances (EPS) involved in adhesion during diatomfouling, we are conducting detailed chemical studies on diatom adhesives. We have isolated andmass cultured three diatoms commonly involved in biofouling, the marine stalk-forming diatomAchnanihes longipes, the freshwater stalked diatom Cymbella cistula (for structure/functioncomparisons), and the capsulc-formning diatom Amphora coffeacforrdis. Purified salk and capsuleEPS has been isolated from these cultures in large quantities, fractionated and characterized bystandard techniques for monosaccharide and methylation analysis. Charged polymers isolatedfrom stalks of Achnanthes differed from those of the freshwater species Cymbella and containboth uronic acid res*dues and sulfate-substituted galactose and xylose units. Residual materialafter water extraction of Achnanthes stalks yielded 3- and 4-linked glucan.

Preliminary analyses of Ampqhora capsular EPS have shown the presence of fucose,galactose, glucose, mannose and xylose residues with complex linkage patterns. In addition, weare conducting culture experiments on the effects of substrate preconditioning on diatomadhesion, including the influence of surface-associated organic matter anid bacteria on diatomattachment, using both uniform organic coatings and natural collections of organic films frommarine locations. These typical organic films will a'so be characterized chemically to providedata necessary to begin modeling the diatom-substrate interaction at the molecular level.

Characterization of Molecular Interactions Between "Conditioned" Polymer Surfacpsand Microbial Biofiln Matrix Polysaccharides

Gill Geesey

Molecular interactions responsible, for the adhesion of microbial biofilms to low energypolymers surfaces in seawater environment are being investigated through NMR and FT-IRspectroscopy of model surfaces, conditioning films and biofilm matrix exopolysaccharides.Silica and germanium surfaces are being coated with n-octadecyltrichlorosilanc (OTS)derivatives to inipart defined hydrophobic or hydrophilic propertiea to the substratum. The OTS-modified germanium substratum surface properties are being evaluated by contact anglemeasurements and attenuated total reflectance Fourier transformi infrared spectrometry(ATK/FT-IR). The decapeptide rep'eating sequence in the polyphenolic protein from the byssalthread of Myti,':;4s edulis is being synthesized and its structure and molecular interactions at theOTS-derivatized surface characterized by complementary cross polarization magic anglespinning 13C solid state nuclea2r magnetic resnance s!ctroscopy (CP!MAS NMR) and

(ATR/FT-IR). Current efforts are focused on synthesis of L-Dopa and 3-OH proline precursors,side chain protection, and FMOC amino group protection for polypeptide synthesis and peptidepurification.

Molecular Characterization of Specific Attachment by a Marine BacteriumDavid Kirchman

Many studiP s have examined the specific attachment mechanisms of pathogenic bacteria.In contrast, little information is available about specific attachment by noupathogenic bacteria.We have been examining how the marine bacterium Vibrio harveyi attaches to chitin, which ison. of the most abundant compounds in nature. Our previous ONR work suggests that thisbacterium uses a 53 kDa peptide to mediate initial attachment. This peptide and another at 40kDa are recovered from chitin after exposure to membrane extracts. The 53 kDa peptide (but notthe 40 kDa) is ov-rproduced by a transposon -generated mutant which exhibits higher levels ofattachment than the wild type. Later stages of attachment appeai to be facilitated by other chitin-binding peptides. In the next phase of our work, we want to explore the roles of these chitin-binGong peptides in attachment.f We hope to obtain E.coli clones that properly express the 53kDa peptide. Additional mutants will swrcened in order to obtain V. harveyi cells defi:ient in the53 kDa and other chitin-binding peptides. These molecular approaches should definitively provethe roles of these peptides in attachment and allow us to begin to examine their regulation.

9

Genetic Responses of Bacteria in BiofilmsA. E. Goodman, M. L. Angles and K. C. Marshall

There is growing evidence that the physiology of bacteria in a planktonic form is verydifferent to that of bacteria existing at a surface, whether loosely or firmly attached to thesurface. The techniques of molecular genetics are allowing us to unravel the modulation andcontrol of the genetic switches that must be occurring to enable bacteria to adapt their physiojogyto changing environmental conditions at surfaces. Biofilms provide an ideal environment forbacteria to engage in gene shuffling, by gen-. transfer mechanisms, and so become geneicallyadapted more rapidly to the environment confronting them. The specific questions that we shalladdress in this paper are:

1. Can a bacterium respond actively to a solid surface?2. Do cells respond activcly to changing conditions within biofilms?3. Within the biofilm does gene transfer occur?

Knowledge about the genetic regulation of such processes will give us a better understanding ofthe. mechanisms of biofilm formation and cell-cell interactions within natural microbialcommunities.

10

DR KURT BAUM DR DAVID KIRC-IMANFLUOROCHEM INC COLLEGE OF MARINE STUDIES680 SOUTH AYON AVENUE UNIVERSITY OF DELAWAREAZUSA CA 91702 LEWES DE 19958(818) 334-6714 (Voice and F.ax) (302) 645-4375/(302) 645-4028 FAX

PROFESSOR GEORGES BELFORT PROFESSOR KEVIN C MARSHALLDEPT OF CHEMICAL ENGINEERLNG SCH OF MICROBIOLOGY & IMMUNOLOGYRICKETTS BLDG 210A UNIV OF NEW SOUTH WALES PO BOX 1TROY NY 12180-3590 KENSINGTON NSW 2033 AUSTRALIA(518) 276-6948/(518) 276-4030 FAX 9-011-61-2-697-2101/9-011-61-2-313-6528 FAX

PROFESSOR TOBY M CHAPMAN PROFESSOR THOMAS J McCARTHYDEPARTMENT OF CHEMITRY DEPARTMENT OF POLYMER SCIENCEUNIVERSITY OF PITTSBURGH UNIVERSITY OF MASSACHUSETTSPITTSBURGH PA 15261 AMHERST MA 01003(412) 624-8250/(412) 624-8552 FAX (413) 545-0433/(413) 545-0082 FAX

PROFESSOR JOSEPH A GARDELLA PROFESSOR KENNETH M MEiZDEPARTMENT OF CHEMISTRY DEPARTMENT OF CHEMISTRYSTATE UNIV of NY at BUFFAL THE PENNSYLVANIA STATE UNIVERSITYBUFFALO NY 14214 UNIVERSITY PARK PA 16802(716) 829-3560/(716) 829-2960 FAX (814) 865-3623/(814 863-8403 FAX

,----,--, GILL GEEY PROFSSO -R GEORGE MUSHRUSHENGINEERING RESEARCH CENTER DEPARTMENT OF CHEMISTRYMONTANA STATE UNIVERSITY GEORGE MASON UNIVERSITYBOZEMAN MT 59717 FAIRFAX VA 22030(406) 994-4770/(406) 994-6098 FAX (703) 993-1090/(703) 993-3193 FAX

PROFESSOR MICHAEL GRETZ PROFESSOR CIHRISTOI'I tER K OBERDEPT OF BIOLOGICAL SCIENCE MATERIALS SCIENCE & ENGINEERINGMICHIGAN TECHNOLOGICAL UNIV CORNELL UNIVERSITYIHAUGHTON Ml 49931-1295 ITHACA NY 14853-" 701(906) 487-202-5/(906) 487-3167 FAX (607) 255-3417/(607) 255-6575 FAX

DR JAMES R GRIFFITH PROFESSOR RALPH S QUATRANONAVAL RESEARCH LABORATORY DEPARTMENT OF BIOLOGYCHEMISTRY DIVISION CODE 6120 UNIVERSITY OF NORTH CAROLINA4555 OVERLOOK AVENUE SW 312 COKER HALl.WASHINGTON DC 20375-5320 CHAPEL HILL NC 27599-3280(202) 767-2210/(202) 767-0594 FAX (919) 962-2098/(919) 962-0778 FAX

DR KYLE D H-lOAGLAND DR JOI IN F RABOLTDEPARTMENT OF FORESTRY IBM AlMADEN RESEARCHI CENTERFISHERIES AND WILDLIFE 650 1 TARRY ROADLINCOLN NE 68583-0814 SAN JOSE CA 95120-6099(402) 472-8182/(402) 472-2964 FAX (408) 927-1271/(408) 927-33M FAX

110

II

Program ManagersPROFESSOR MICHAEL L SLNNOIT Dr. Randall S. AlberteUNIV OF ILLINOIS AT CH-IICAGO Molecular, Cellular & Environ. BiolPO BOX 4348 Office of Nava' ResearchCHICAGO IL 60680 800 North Quincy Street(312) 996-63101/(312) 996-0431 FAX Arlington, VA 22217-5660

703) 696-4039/(703) 696-1212 FAXPROFESSOR J HERBERT WArTECOLLEGE OF MARINE STUDIES Dr. Harold E. GuardUNIVERSITY OF EELAWARE Polymer and Organic ChemistryLEWES DE 19958 Office of Naval Res.alch(302) 645-4257/(302) 645-4007 FAX 800 North Quincy Street

Arlington, VA 22217-5660PROFESSOR DAVID WHITE (703) 696-4311/(703, 696-2611 FAXCTR FOR ENVIRON BIOTECH10515 RESLARCH DRIVE SUITE 300 Dr. Michael T. Marron

UNIVERSIIY OF TENNESSEE Molecular, Cellular & Environ. BiolKNOXVILLE TN 37932-2567 Office of Naval Research(615) 974-8030/(615) 974-8086 FAX 800 North Quincy Street

Arlington, VA 22217-5660(703) 696-4033/(703) 696-1212 FAX

Dr. Kenneth J. WynrteL uiymerIULU %r-aruc 11UL 1-1ientLiv

Office of Naval Research800 North Quincy StreetArlington, VA 22717-5660(703) 696-4409/(702) 696-2611 FAX

CAPT Stephen SnyderNaval Research LaboratoryWashington, DC 20375-5320(202) 767-3115/(202) 404-7139 FAX

12

Grant No.: N00014-93-1-0491

DISTRIBUTION LIST FOR REPORTS

ADDRES,1E NUMBER OF COPIES

Scientific Officer Code: 1141MB 3 copies of proceedingsRandall S. AlberteOffice of Naval Research800 North Quincy StreetArlington, Virginia 22217-5660

Grant Administrator 1 copy of proceedingsOffice of Naval ResearchRESIDENT REPRESENTATIVE N66005THE OHIO STATE UNIV. RESEARCH FDN.1960 KennyRoad RM 113Columbus, Oi 43210-1063

Defense Technical Information Center 1 copy of proceedingsBuilding 5, Cameron StationAlexandria, Virginia 22304-6145

HI

-FROM UNC-CH CONTRACTS C GRANTS 09.13,1994 15i14 2 P. 21~Fo'rm ApprOvadREPORT DOCOMENTATION PAGE.I _____________________ OM.ft. OIO4 -0188 -

PI~C bW- t h k 0 m m'etlo Of 1AW1141eif It ..IJMtt.- to aweM69 I IKOW W IEA1AW. IntfIudIng tiptwist foe MOMIW IfauuClons ~ ngeis t

I7AmNCV 11.1ftUY (Leuv W1nJ~~ ORT 5AT1i RPYTPE AND'~ D1 COVERE

4.r V~L APDBIT&I5. FUNDING NUMBERtS

Molecular Interactions at Marine Interfaces IN00014-93-1-041~9

6.rLjHM-

Ralph S. Quatrano

7. pinfolimI 79FGANIZTIoN APAFS ASD ADORESS(ES) S. PFAFORMING ORGANIZATiO0N_Univ. (f North Carolina at Chapel. Hill REPORT NUMBERDepartpent of Biolog-y

E` GB #32W0 - Coke;- hfalEll Chapel Hill., NC 27599-3280

li. SPONSORINGIMON11OWNG AGIN". NIAMI(S) AND ADDRI SSRS) 10. SPONSORING tMONi rOUNGContra ts and Grants 1AGtWCY REPi'4, NUM5LIR

440 W. Franklin StreetGB #1130Chapel Hlll, NC 27599-1350

11. SUPPLEME tAR7 NOTES -

Worksho Ischedule, abstracts and list of partLcJi-parits attached.

12S. OISTRi3U1It6N/AVkIILA8ILTY STtMIENT 12b. DISTRIBUTION CODlE

tinlimiled Unlimited

11AIS~T ACT (Ma)xImUm 2ýOwords) . -

Approxim t~ely 25 scient~ists frU.1- around the world Tnarticipated in a workshopajimed at discussing molecular interactions at mairine interfaces fromn twope rs ?c t -v(2s: the chemnistry of adhe.sive polymers and the biology of st ructuresthat secr-e as att~achlinent organs of marine organi.'ýiis. Detailed prSeseraatioiý3 ofeach area was comxplCineInted by jo1int discussions Of theC two persrneC~Lves iti orderto achieve jint2raction on the research p)rogranis supported by ONR.

TV it-Clr 15. NUM8(R OF PA'TEl

Adhiesi in, biofouling, adnesive polymers Z'

MUM IO IIII. SZURtITY CLAWSFICATION 19. SECURITY CLASSIWICATIOIN 20.1LIMITA-TIUONFAUSINAr.Tor RIpoi Y ~ OF THIS PAGt. Of AP$TRACT ~I

r~t 7 !41 Iý :i; ii 5 Stl:rndeed Form 29t3 (R"v. 2 09774NCK~iiI* by AN'SI lid WillIMu VW