Microbial symbionts of Great Barrier Reef sponges

14 Pages·1998·5.3 MB·English

by  A M Burja

I
MICROBIAL SYMBIONTS OF GREAT BARRIER REEF SPONGES
>AM M. BURJA. NIC OLD S. WEBSTER. PETER I MURPHY AND RUSSELL T. HILL
BUrja, A.M.. Y\'ehster. N.8., Murphy, P.T. & Hill.R/I 199906 30: Microbial Svmbiontsof
GreatBarrierReefSponges.Memo'u5o/'//ycQueewlfru.lMuseum44:63-75.Brisbane. ISSN
9079-3835.
Microbial symbiontsoj rw«i sponges, Rhopaloekiesodorahik(Oietyoceratida: Spongiidae)
and a new species 'Yen White Fan1 (VWF)(DTcfyoceratvda: Phyllospongiidae), Lire being
studied in detail. Bacteria isolated from A', ud&wbile, V\VT. and the surroundingarribienl
seawater were characterised using morphological, biochemical, arid molecular techniq
Inthe case ofIt odurahilv, a single bacterium., designated NW001, was found todominate
ulturablebacterialcommunity associated with the sponge but wasabsent fromambient
seawater samples. Strain NW00I was predominant in aJl individual sponges sampled
(N-40) from different regionsot theGreat BarrierReef, generally at more than an orderof
magnitude greater than the second most common bacterium (NW002). The bacterial
community associated with & adarabfkappears tobeflighty stable. InthecaseofVWF, ihe
.irable bacterial community was more diverse and showed greater variation between
individuals. This community general!}- comprised eight predominant bacteria, rarely
isolated from water samples and constituting ca. 70% of the total eulturable bacteria.
I.viensi\e biochemical testingwasperformedona!! isolates*togivedata forclusteranalyses
to identify the major groups of bacteria present. One isolate from each sponge was
characterisedat the molecular level by PCRamplification and sequencingof!6S ribosomal
RNA gene fragments. Analysis of sequence from NW0D2 indicates it is a
Pseudoattemmouus sp; Strain E300043J5 from VWF is a mteroalga. with 16S rRNA
sequence from its piastidcloselyrelated tothat ofother plastirfs nfrntfrine eukaryotk algae.
Thisstudy produced anarraj r>fwell-characterised Enjctobes fOJ natural products ser-
in paru'. inn far importantcompoundsknown tobe protiuc ffisfH
Sponge, syuihioHS, Djctyoceratidff, Demospon I6S rRNA. Rh lias adarah
hiirmaisiiw, Vibria,
i.dfeemniceM,lUPiMrLhi:So P3. TioMwunrspvihfyl,e\MfGa,rlnOeLBiDopr4a8dIuQetA&uGsrtroaulpisa:AuNsitcroalleianWeIbnssttietru,teFoajc'uMlfftryjrto/je
Health, Lite, and Molecular Sa&iCoz, Jam tft University ofNorth Queensland
txwnsviUc. OLD 48 /, Australia; Russell 7. Hill (email: hillna urftbl uwdedit), Cento, vj
Marine Hioteelmotogy, Umversit] of Maryland Biatechnotbi titute 01 East Pratt
. . Buttmtort: MD 21Jul USA: 4April I
Symbiosis isconsideredapermanentassociation matrix and can occupy up to 60% ofthe sponge
between organisms ofdifferent species. This term volume (Wilkinson, 1978a).
is not restated to rnutualistic associations but The HoIm£V ofbaUcnum_sponuc associations
encompasses all associations, regardless ot the hasd]CUed considerablc intcrest ^(m researchers
type oj interaction between the individuals. investigating novel chemicals derived from
Mutualistichactenal-mvenebratesymbiose^have sponges. The term symbJonl has been broadly
been reported from many invertebrate taxa. applied and few investigators have explored
Examples ofthese meltidc cellulolytic nitrogen metabolic relationships and capabilities of the
fixingbacteria fromwoodboringbivalves(Shieh symhtont-host complex. One approach that will
&
Lin. 1994).methanotrophicbacteriaofbivalves contribute to understandingtheserelationships is
(Dubilier et aL, 1995) and bacterial syrnbiotltS pf to isolate symbiotic bacteriaand investigate their
echinoderms (Burnett & McKerizie, 1997; Kelly metabolic and taxonomic characterisi
& McKenzie, 1995). Although chemoautotrophie Cosmopolitan microbial wmhiont* associated
symbiosis has received the most attention, there vyith marine sponges include heterotrophic
are also many symbtoses where the type ot bacteria, cvanobactcria and unicellular algae,
interaction between the host and then symbiont Numerous studies have described three general
remainsunknown. Mostsymbiotic bacteria from classes of heterotrophic bacterial-sponge
spongeshave been located withinthe intercellular associations (Wilkinson. 1978a). 1 ) Small
1
64 MEMOIRS OF THF QUEENSLAND MUSEUM
populationsofuosmppdlitanbacteriawiffiaspecies MATERIALS ANDMETHODS
composition similar to thai (bund in the ambient
iu_t These are most likely utilised asa food iNGE COLLECTION AND BACTERIAL
rcc by the sponge. 2) Species- specific popul- ISOLATION. Material examined in this study
ation inhabiting themesohyl region, not found in was collected using, SCI I M)m depth").
seawater, and most likely comprising true Seasonal sampling for bacterial community studies
symbionts. 3] Fairly ill-defined, consisting of was conducted o\ I months at Davies Reef
liketleyriatolobceatetdruewitshyimnbitnhnetsS.pOPHhenoty; pH (ASuOstnraaultiicaa,l milEeSs°4of9f.6TovS\.ns\ i|ll4e7, cO3ue4e.n4s91
iclared bacterial symbionts have been describe!d Immediately after collection, specimens
ii onomicaily disparate sponges collected processed for bacterial isolation. Using aseptic
raj ally remote Iocj technique, 3 km" section ofsponge tissue
Sponge syinbioni ls have included members I>1 'lie ehxocmiosegdemansdedsuirnfasctee-rsitleerairltiisfeidc.ialSpsoenagweattiesrsu(eASWl
ecus and using a uv.-riarand pestle. Serial dilutions
|
W&tftt (Sanlavy el al , 1990). U has boon I0*1 and I re prepared in ad plated
determined that t'acullarivt i: onto several media for isolation ofmicrobes.
n.iiahvthsi- a wide- nmge ofciompounds and may. Media for isolation of heterotrophic ba,
imipjoi1r1atIteasnhtaarsienanrloestomeoib.receulnatpionsgtuwlaatiteerd(L\tuVChtaU:tfcisntIinciksyi were1roUnpsoanet-dlsieionlgetechntiiiscvsetvuimdbyar.rioiDsnifef(eQmoxeoMdtaidr)uimnaenAdPgCaSBrBS2A2.f1o6ia
;oIoniea may wtribiitors Ctive medium for bacterial pathogens (Oxoid
1
XirjufguencSttriuocntsurtahlairighiadivtey tbWeielnkisnusgogne,st|e9d78c)« af. 1i9m7b1ia) aBnlid MN + tBr1B2as(eW).aiBcGrb-ulry1 (&StaSrtifii
-.ymhunits include digestion of 1978) were used for isolation of oxygenic
tg the !i( ' i tfi . Litect phototrophs. Plates were incubatedat27
uporatiaa ofdissolved organic matter from period ranging between 2- Total cultur-
rates,anddigestionandrecyclingofin i I 1 1 ci tony counts were obtained from I »if
uble sponge collar Man' 1216spreadplates. Representati1
i
poitgc syinbionls are of biotcchnoiogical ach morphotypc were cultured Irom initial
interest since bioacliee compounds ofpotential isolation and cryoprcserved tor further stt*
medical importance isolated Irom sponges Total bacterial counts were deterno ted
'in origin(Bewfcy& Faulkner, 19, 98; fluorescent microscopic, enumeration of ceils
stained with 496-diatntdino-2-phetivImdole
ley ei af. 1996; Stierle et af. L988) 1 Here bedby Porter & Feig(198i
intakes in
Symbionts winch produce bioacti\e compounds, ANNING AND TRANSMISSION
lading consistent yield and large-scale ELECTRON Mb :'PV. Sponge sec
production in iermemers, obviatingthe need for were pre I blade to Cut
collection of sponges Irom natural cc Jmm thicksectionsofsponge'tissue, ensuring
l/ilinskaset af, 1995). tha1t both the cciosome and choanosome were
In this study, microbial symbionts v represented. Sections were fixed in 2
investigated in twfl Great Barrier Reef sponges, glutaraldehyde made in 0. 1 M sodium caeodylak-
Rh<>; tes odorabiie Thornpson et at. butler fur ZOlirs. fixed samples wci
(Dictyoceratida: Spongiidae) and a new species. into 1 1 odium eacodylate and stored aj 4°C
ted here vVery White Fan' (VWF) (sec until further processing. Sect ponce
jqirist et al., L999, this volume). This is a first tissue were placed in a 1% osmium tetroxide
ascertainingwhether these symbionts solution (prepared in D.2M potassium phosphate
Ii ed in the production id important buffer,pll 7.4)tor3.5hrs, Sections were pexQi
ctive compounds tes odombite., and washed thoroughJ> in sterile distilled writer,
common throughout ihe GBR, produces novel dehydrated in aexactedcfhano series! 15%
I
norscsiertcrpenes (rhopaloie actdsi whicll exhibit 75%, 85%and " aced in embedding
,, 1
potentcytotoxicactwinestOhtaet and capsules and coveted with Spurr's resin. Thin
>" contains the compound fanolidI eI {if sections were cut and stained with 2% ui
Murphy, unpublished data), which retards the acetate followed by 0.2% lead citrate.
growth ofseveral tuniourccl] lines. were mounted on 200 mesh copper TLM grids
GBR SPONGE MICROBIAL SYMBIONTS 65
TABLE 1. Type cultures used as control strains in morphological characteristics were determined:
biochemical analyses. Key: ACMM, Australian colony morphology, gram stain and cell
Collection of Marine Microorganisms; ATCC, morphology, plate swarming, oxidation/
American TypeCultureCollection.
fermentation(Leifson, 1963; Lemoselal., 1985),
Collection number Organism oxidase, catalase and growth at various salt
ACMM667 Vibrioparahiwmoiviicus concentrations (0%, 1%, 6%, 8%). In addition
AACCMMMM68698 VV..ipbarnio/haategimnoolivyltiiccuuss s1e5v0e|riagl,aanmtpibiicoitlilcinsus1c0e|pitgib&ilitpyolteysmtisx(i0n/1B29510iu&)
ACMM90 V parahaemolyticus were performed along with growth on different
media (Lecithinase^ DNase, TCBS, SBA).
ATCC33809 Vibriofhivialis Several American Type Culture Collection
ATCC33807 V. fluvialis (ATCC) and Australian Collection of Marine
ATCC 7966 Aeromoruishydrophila Microorganism (ACMM) strains were included
ATCC35624 Aeromonasgroup77 as controls (Table 1 ). Isolates were tentatively
ATCC33509 Vibrioordalii identified to either the genus or species level by
comparing their phenotypic characteristics with
coated with carbon and Formvar. Samples were those of type cultures and by comparing
visualised following standard scanning and biochemical test results, carbohydrate utilisation
transmission electron microscopy techniques at patterns,andcellmorphologiestothoseofspecies
James Cook University and University of described in Sergey's Manual of Systematic
Queensland. Bacteriology (Holt, 1986) and Bergey's Manual
of Determinative Bacteriology (Buchanan &
BIOCHEMICAL TESTING OF BACTERIAL Gibbons, 1974).
ISOLATES. The isolates were characterised by DATA ANALYSIS. The levels ofrelatedness of
determiningbiochemical characteristics in 96-well the bacteria were determined from the pheno-
mHiacnrosteintr&e-pSloatrehseibmas(e1d99on1 ).mSeetvheordasl ddyeescirnidbiecdatboyr t1y9p8i4c).data using Jaccard's similarity index (Zar,
toersntisthwienre,ebpaesref;onrimteradt:erMeodlulcetri'osn,arOgNinPiGne,, ilnydsoilnee,, Sy =a/(a + b + c)
acetoin,tellurite,aesculin,alginate,acid-arabinose, whereSj=Jaccard'ssimilaritycoefficient, a=no.
arbutin, glucose, inositol, mannose, salicin, species in sample A and sample B (joint occurr-
sorbitol, sucrose, and urea. The following tests ences), b = no. species in sample B but not in
wereperformedtodeterminetheabilityto utilise sample A, c = no. species in sample A but not in
different carbon sources in assimilation broth: sample B.
arabinose, cellobiose, galactose, glucose, Phenograms were constructed by using
mannose,melibiose, lactose,melizitose,sucrose, unweighted pairgroup mean average (UPGMA)
trehalose, xylose, ethanol, glycerol, propan-1-ol, linkage (Sokal & Michener, 1958), Euclidean
sorbitol, gluconate, glucuronate, amygdalin, distances and the computer software package
arbutin,citrulline,hydroxyproline,leucine,gluco- STATISTICA (StatSoft Inc., Tulsa, Oklahoma).
samine, hydroxybutyrate, a-ketoglutarate,
succinate, base (control), adenine, aminovalerate, BACTERIAL IDENTIFICATION BY 16S
N-acetyl-D-glucoseamine, ethanolamine, RIBOSOMAL RNA (rRNA) ANALYSIS. Two
m-erythritol, D-fruetose, D-galacturonate, microbial isolates, NW002 from R. odorabile
glutarate, inositol,malonate,maltoseandvalerate. and the microalga E30004315 were identified
The assimilation broth contained (per litre) using a molecular approach, partial sequencing
0.015g of yeast extract, l.Og of ammonium ofthe 16S rRNA gene fragments amplified from
chloride,0.075gofdi-potassiumhydrogenortho- these isolates using the polymerase chain
phosphate, 6.1g of Tris (hydroxymethyl) reaction (PCR). Total DNA was prepared from
aminomethane and 15g ofASW salts (pH 7.5). strains NW002 and E30004315 using a method
Afterautoelaving, the carbon sources were filter modified from Ausubel et al. (1987). Oligonucleo-
sterilised and added aseptically to a final tide primers with specificity for eubacterial 16S
concentration of8%(wt/vol.). Inoculationswere rRNA aenes [Forward primer 8-27:5\AGAGTTT
performed by suspending colony material in GATCCTGGCTCAG -31 (modified from FD1)
ASW
and inoculating lOOuJ of this suspension (Weisbunretal., 1991)andReverse primer 1492:5'-
into each test. In addition, the following GGTTACCTTGnACGACTT-3, (Reysenbach et
66 MEMOIRS OF THE QUEENSLAND MUSEUM
al., 1992)] were used to amplify a
OS rRNA gene fragment from
I
NW002. The cyanobacterial and
plastid-speeific 16S rRNA primers
described by Niibel cl ai. (1997)
were used "tor E300043I5, since
thisisolate, althoughunialgal,may
have been contaminated with low
numbers ofheterotrophic bacterid
PCRfragmentswerepurifiedusing
the Microcon 30 system (Amicon,
Beverly. MA), and sequenced
using the PRISM Ready Reaction
Kit (PE Applied BioSystems.
FosterCity, CA) and an AB1 310
sequencer PE Applied
i
BioSystems). Sequencingdatawere
.cd by comparison to los
rRNA genes in the Ribosomal
Database Project {Maidak et al..
1999; Maidak et al., 1991) and the
(ienbank database, and aligned
manuallyusingthePhydiisoftware
(Chun, 1995).
EVoLutiotiary trees wcrc
inferred using the neighbour-
&
joining (Saitou Nci. 1987).
Fitch-Margoliash {Fitch &
Margoliasli, 1%7)andmaximum-
parsimonv (Klu^e Si Harris.
1969) algorithms in the PI1YL1P
package (Felsenstein, 1993).
Evolutionarydistancematrices lor
the neighbour-joining and Fitch-
MargoIiash meIhods werc
generated as described by Jukes
& Cantor! 1969), Tree topologies
were evaluated by performing
bootstrap analyses of the
neighbour-joining data, based on
1000 re-samplings (Felscnstein. I'Ui 1. Electron micrographs of VWF sponge sections. \, Low
1985).
Abbreviations: A IMS, magnificationscanningelectron micrograph oJ spongesection. U,High
magnification ofsponge section showing cells identified as putative
Australian Institute of Marine eyanobaeteria on morphological criteria, ( -D, Low and high
Science; ASW, Artificial seawatcr; magnification, respectively transmission electron micrograph of
IMP1. Diamidino-phcnylindolc. sponge mesohy] section showing location of bacteria within
16S rRNA, 16S ribosomal kbactcrioc\tcs' and putative eyanobaeteria, indicated by arrows.
ribonucleic acid; SBA, Sheep
Blood Agar; TCBS, Thiosulphatc Citrate Bile electron microscopy to be present within the
Salts Medium; VWF, Very While Fan. sponge VWF {.Fig. IA-D). Bacteria closely
associated with the sponge tissue, possibly
RESULTS embedded in a polysaccharide matrix, were
presumed to be eyanobaeteria based on morpho-
ELECTRON MICROSCOPY \ large and logical crileria (Fig. IB), since these cells
complex bacterial community was shown by resemble filamentous eyanobaeteria (e.g. genus
.
GBR SPONGH MICROBIAL SYMBIONTS
f>7
OsaVatoria). Cellspresumed to
beothereubacteria, basedonthe
standard morphological criteria
of size, shape and membrane
structure, were also in close
contact with the sponge tissue
and contained in cellular
organelles resembling the
"bacterioeytes1 described by
VacelctA Donadey 1977)(fig.
(
C-D). Sand grains were
1
observed which appeared to be
incorporated into the aponge
external structure (also reported
by Bergquist, et al., 1999),
possibly performing the function
of increasing structural integrity
(Shaw, 1927).
The bacterial community
within R odorabile was also
large and appeared to be
comprised oi' many different
bacteria (Fig. 1I. The bacteria
appearto bedispersedthroughout
the sponge mesohyl and no
bacteriocytes were evident. In
contrast" u> VWF, cells FIG. 2. Transmission election micrograph showing the diversity of
resembling cyanobacteria were bacteria]morphologiesand thedensityofbacterialcellswithin thetissue
not seen. ofthe sponge R odorabile.
BACTERIAL FNUMI•RATION VWF individuals from different locations
Tlte average number of eulturable bacteria from
VWF revealed several similarities. Eight eubacteria,
direct plate counts obtained from was designated AB001 to AB00S. were frequently
3.6x10 /ml and the average Lota! count observed
from0APJ stainingwas6.3x1oVnii.Only 0.0<v:n cObosmemruvneidtaysobfeVinWgFparatndofwtehreeefuolutnurdatbolebebapcrteesreinatl
oftotal bacteria were able lobe recovered using
traditional culture techniques. The range oftotal only in small numbers in samples from the water
column.
and eulturable bacterial counts found in samples
fromeightindividual VWFspongesareshownin Atotal of220 isolateswereisolated lrom VWF
Figure 3, samples collected between June 1997-May 1908
The average percentage ofeulturable bacteria from locations between Trunk Reel'and Davies
0f.r0o0m1A-\0.o8d%o.raTbhieleawvaersaognelypeQr,c1e%ntawgiethoafrbaanctgeerioaf Rceoenff,ormGerdeatto oBnaerorfieerighRteeclfu.steTrhse(sFeig.i5s)o.laTtweos
able to be cultured from the water column was clusters were Gram-positive with the remainder
0.23% with a range from 0.003-0.9%. Total and being Gram-negative. The Gram-positive
eulturablebacterialcountsfoundinsamplesfrom bacteria were further sub-divided by means of
four individual A\ odorabile sponges and the cellular morphology. Approximately 40% ofall
ambient seawater surrounding each sponge are bacteria (including strains AB004, AR007, and
shown in Figure -1. AB00S)isolated from VWFclustered in phenons
6 and 7 (Fig. 5); these phenons contained the
BIOCHEMICAL CHARACTERISATION Of vibrio and aoromonad representatives, re-
BACTERIAL ISOLATFS. Morphological and spectively, ofthe ribrionaceae type strains used
biochemical dataindicatedthat,at leastasjudged m this study. In addition, approximately 30% oi
lrom the eulturable fraction, the bacterial the total bacterial community fell in a single
community within VWFdiffered from thatpresent phenon (phenon S), which contained the strain
in the water column. Culture results lrom 15 AB005, Of the remaining three Grani-ne&aihe
68 MEMOIRS OF THE QUEENSLAND MUSEUM
1.0C&08
I 1.0C&07
—
&1.00B06 -I I
^
I I
§ 1.00&05
2 1.00BO4
§ 1.00B03
o
FIG. 4. Total and culturable bacterial counts from
FIG. 3. Total and culturable bacterial counts from tissue offourR. odorahiie individuals and seawater
tissue ofeight VWF individuals collected at Davies surrounding each individual collected at Davies
Reef, Great Barrier Reef. Reef, Great BarrierReef.
clusters,phenon3 containedpigmentedbacteria, catalase-positive and separated on the basis of
phenon 4 included strains AB003 and AB004, carbonsourceutilisation. Oneofthefiveclusters
and phenon 5 included strains AB001 and containedsmAeromonassp.typeculture(phenon
AB002. StrainAB001 appears closely related to 6) and a second cluster contained the Vibrio
NWOOl fromR. odorahiie*withbothstrainsbeing anguillarum type culture (phenon 4). NW001
representatives ofthe alpha-Proteobacteria (data was a Gram-negative rod; oxidase, catalase,
not shown). urease, VP and indole positive; utilised adenine
From biochemical and morphological dihydrogenase and had the ability to utilise
observations, it was apparent that the bacterial glucoseandgluconateascarbonsources.NW002
communitywithinR. odorahiiewasquitedistinct was a Gram-negative rod, oxidase, catalase, VP,
from the bacterial assemblages associated with indoleandacidarabinose positive. Itwas urease—
the ambient water column. In general, both total cneagrabtoinvesaounrdcedsi.d nBootthutiNliWse00an1y aonfdthNeWt0es0te2d
and culturable counts from sponges exceeded
counts from the corresponding water samples. clustered within phenon 5.
oTrhgeansipsomngedemsiicgrnoabtieodtaNwWaOsOld,omiwnhaetreedasbythains MICROALGAL ISOLATES. In addition to the
isolate was completely absent inthe surrounding heterotrophic bacterial isolates, eight straiVnsWoFf
water column. A small component of the oxygenic phototrophs were isolated from
microbial community was observed in both the wanadsocnhearoafcttehersiesesdtrabiyns,1d6eSsirgnRaNteAd Ese3q0u0e0n4c3i1n5g
sponge tissue and the ambient seawater. (below). A single phototroph strain was isolated
A total of223 isolates were collected from 40 from R. odorahiie. Phototroph strains were not
R. odorahiie samples collected between August characterised by biochemical testing because of
I997-May 1998.Theseisolatesconformedtoone the difficulty in identification ofmicroalgae by
of ten major clusters (Fig. 6). Two clusters this means; instead I6S rRNA sequencing was
(phenons 9 & 10) were Gram-positive and these used as a method for identification of strain
were distinguished from each other on the basis E30004315.
of the oxidase reaction. Two of the Gram-
negative clusters (phenons 1 & 2) were oxidase- PHYLOGENETIC POSITIONS BASEDON 16S
negativeand showed profiles linkingthem tothe rRNA SEQUENCING. Phylogeneticrelationships
Enterobacteriaceae. One of the Gram-negative, fortheplastidofmicroalgaE30004315 from VWF
oxidase-positive clusters (phenon 3) was catalase- andheterotrophicbacterial strainNW002 from R.
negative and the remaining five clusters were odorahiie are shown in Figures 7 and 8,
5
GBR SPONGE MICROBIAL SYMBIONTS 69
a
2
2
1 Grampositiverods Streptococcusspp.
7 Grampositivecocci UNIDENTIFIED GROUP
3 Gramnegativerods PIGMENTEDBACTERIA
A Gramnegativerods UNIDENTIFIED GROUP AB003& AB006
5 Gramnegativerods PROTEOBACTERIA AB001& AB002
g Gramnegativerods Vibriospp. AB004.AB007&AB008
•7 Gramnegativerods Aeromonasspp.
Q Gramnegativerods Photobacteriumspp. ABQ03
FIG. 5. Similarity dendrogram for isolates obtained from VWF.
respectively.Theplastidfrom isolateE300043 invertebrates. Several studies have documented
VWF 1
irom is closely related to plastids ofother diverse microbial communities associated with
marine eukaryotic algae. NW002 is a Pseuch- sponges (Vacelet, 1970, 1975; Vacelet &
alteromonas sp. Donadey, 1977; Wilkinson, 1978a,b,c; Santavy,
1985; Willenz & Ilartman, 1989; Santavy &
DISCUSSION
Colwell, 1990; Santavy et al., 1990; Lopezetal.,
1999). These communities are generally
Thiscomprehensivebiochemical and morpho-
logical analysis of bacteria isolated irom two comprised of large numbers of heterotrophic
Great Barrier Reef sponges further emphasises bacteria thatoften occupy upto60%ofthesponge
thevariability inmicrobiotaassociatedwithmarine volume (Santavy, 1985; Wilkinson, 1978a,b).
70 MEMOIRS OF THE QUEENSLAND MUSEUM
2.0
1.5
Q^h
1.0
0.5
mm
0.0
i Gramnegative, oxidase(-) Snterobacteriaspp.
2 Gramnegative, oxidase(-)
'i Gramnegative, oxidase(+), catalase(-)
A Gramnegative, oxidase(•*-), catalase(+) Vibriospp.
flj Gramnegative, oxidase(+), catalase(+), CSU (-) NW001 & NW002
Gramnegative, oxidase(+), catalase(+), moller's(+)
f:
"T Gramnegative, oxidase(+), acid(+)
© Gramnegative, oxidase(+), acid(-)
»<
*< Q Grampositive, catalase(-), oxidase(+) Streptococcusspp.
1 A Grampositive, catalase(-), oxidase(-) Streptococcusspp.
FIG. 6. Similarity dendrogram for isolates obtained fromR. odorabile.
PhenotypicanalysisofbacteriafromtheCaribbean evident from the present study, that the sponges
VWF
sclerosponge, Ceratoporella nichohoni revealed Rhopaloeides odorabile and support
significant differences in sponge and seawater taxonomically diverse microbial assemblages,
phenotypes (Santavy & Colwell, 1990). It is High microbial diversity is not surprising when
GBR SPONGE MICROBIAL SYMBIONTS
71
MarinecloneHstpLl reported that many sponge phototrophs are
—
morphologicallyflattenedtoincrease surfacearea
82.f. p —MarinecloneHstpL4 for interception oflight. This is consistent with
StrainE30004315 the structural morphology ofVWF. In contrast,
only a single phototroph was isolated from 7?.
Rhodophyte plastidEnvOCS54 odorahile and cells with characteristic
cyanobacterialmorphologywerenotobservedon
CjanobactenuwcloneLD27 microscopic examination ofR. odorahiletissue.
Ansptew—McdLsimchloropiast The culturable bacterial community of R.
r odorahilewasdominatedbystrainNW00 which
100.f.p i SternacwfttacMoroplatf comprised 74% of the total culturable b1a,cterial
community in this sponge but was consistently
L 5Up *-Metowmpeudocosfohtmchloropiast absentfromtheseawatersamples.Thisisthefirst
-|—sup Marineeubacteritimagg56 report of a single bacterium comprising such a
OkMkmrm high proportion ofthe culturable bacteria from a
1 chloropiast sponge. Previously, aspecific bacterial symbiont
0.01 was found in nine often sponges oftwo classes
1 /tau/atdirtcohchloropiast and seven orders, and a second symbiont was
specific to the sponge Verongia, but only as one
FIG. 7.Neighbour-joiningtree for631 bpofsequence componentofalargemixedbacterialcommunity
poKbretiyam:ienrfesadnfdruospminisgntdrciaicynaatnEeo3bba0rc0at0ne4crh3iea1sl5taihsnaotdlawtpeelrdaesftiardlo-smsopVefcWoiuFfni,dc b(eWtiwlekeinnsNoWn0e0t1ala.,nd1R9.81o)d.orTahhielereprloavtiidoensshainp
using the Fitch-Margohash or maximum-parsimony ideal model system for investigating the
methods, respectively. Thenumbers at thenodes are relationshipbetweenthisstrainanditshostsponge
percentages(onlyvaluesover50%shown)indicating because this isolate is predominant and has a
the level of bootstrap support, based on a characteristic colony morphology which
neighbour-joininganalysis of 1,000 re-sampled data facilitates enumeration of strain NW001 based
sets. Scale bar represents 0.01 substitutions per solely on colony morphology. Initial indications
nucleotide position. are thatthisrelationship persists overspatial and
temporal scales and is highly stable (work in
considering that sponges derive their nutrition progress). Although the relationship between
from filtering the ambient seawater. It has been NW002 and R. odorahile appeared less stable,
demonstrated thatmarine sponges arecapableof NW002 was frequently the second most
discriminating between food bacteria and predominant culturablebacterium (afterNW001)
bacterial symbionts. The mechanisms for this present in R. odorahile, and was present in the
recognition are not clear but it has been spongetissue atmuch higherconcentrations than
postulated that sponge phagocytic cells do not detectable in the ambient water surrounding the
recognise the capsule coating of symbionts sponges. Similarly, strains AB001-AB008 were
(Wilkinson et al., 1984). consistentlypresent inthespongeVWFathigher
Eightpredominantheterotrophicbacteriawere concentrations than detected in the surrounding
evident in the culturable community isolated seawater. The mechanism whereby the sponges
from VWF and these isolates were generally acquire these symbionts is a topic of current
absent from the surrounding seawater samples. research.
These culturable bacteria clustered in several It appears as though the two sponges adopt
phenonsonbiochemical analysis. Theculturable different strategies for harboring their microbial
community ofVWF was more diverse than that communities. Rhopaloeides odorahilemaintains
observed in R. odorahile and showed greater the bacterial ceils in the loose matrix of the
fluctuations between individual sponges. One mesohy1,whereasVWFappearstoincorporatethe
notable feature was the prevalence of cells cellsintostructuresreferredtoasbacteriocytes. In
resemblingcyanobacteriawithintheVWFmatrix, VWF,thebacteriaarealsocloselyassociatedwith
observed by microscopy. Also, eight strains of thesandgrainsjustbelowthecuticle.Thereasons
phototrophs were isolated from this sponge. It is for these different approaches are uncertain but
postulated that VWF is a phototrophic sponge, may relate to the structural composition of the
deriving a component ofits carbon budget from sponge orthe function ofthe bacteria within the
photosynthetic symbionts. Wilkinson (1992) has spongetissue.Rhopaloeidesodorahilemaintains
72 MEMOIRS OF THE QUEENSLAND MUSEUM
AUerotnonasaurantia Gram-positive bacteria, which made up
100fp approximately 10% ofthe total isolates in each
Alteromonascitrea case.Earlystudiesofmarinemicrobiology found
Psevtdoalteromonassp. ANGjo2 that about 95% ofmarine isolates were Gram-
-negative (ZoBell, 946)butrecently30%ofthe
'-: r 1
Pseudoalteromanassp.MB8-11 bacteria associated with a marine alga were
&
L_MI5S67ff.pHydiothemmlventstrainPVEOTU5 f1o9u9n5d) taondbeitGriasm-lpikoesliytitvheat(tJheensepnroporFteinoincalo,f
NW002 Gram-positive bacteria in most marine habitats
I &
hasbeenunderestimated(Jensen Fenical, 1994).
^—NorthSeabacteriumH? Gram-positive bacteria include actinomycetes, a
76f.p —Ps&vtdoalteromonassp.SWOS dgirsocuopveorfyp.articular importance in natural products
tPseudom.on.asatlantica Because of the difficulties in accurately
S8f.p identifying marine bacteria by conventional
•Pseudoa.iterom.anashaloplcmktis
biochemicalcharacterisation,moleculartechniques
^ Pseudoaltarom.onasgracilis are the most appropriate for unequivocal identif-
83f ication of marine bacteria. A single isolate
Pseudoatteromonascitrea (NW002) fromphenon 5 oftheassemblage from
Pseudoaiteromonasantarctica R. odorabile was selected to demonstrate the
'"-Lif utility ofthis approach and as a first step in the
Alteromonasetyxkavn molecularidentification ofone isolate from each
87f^ phenon. In addition, because ofthe difficulty in
Pseudoalteromanasnigrifaciens identification of marine microalgae by
63fl
conventional techniques, phototroph isolate
01 Alteromonasdistincta E30004315 was characterised by 16S rRNA
FIG. 8. Neighbour-joining tree for over 1,000 bp of sequence analysis ofits plastid.
sequenceobtainedusingeubacterial-specificprimers Sequence analysis ofthe plastid ofmicroalgal
fromstrainNW002 isolatedfromR. odorabile.Key:f strain E30004315 revealed that this plastid was
andpindicatebranchesthatwerealsofoundusingthe
Fitch-Margoliash or maximum-parsimony methods, most closely related to sequences of clones
respectively. The numbers at the nodes are HstpLl and HstpL4, cloned from a library ofthe
percentages(onlyvaluesover50%shown)indicating unculturedmicrobesassociatedwiththeseagrass
the level of bootstrap support, based on a Halophilastipulacea,aubiquitousseagrassfrom
neighbour-joining analysis of 1,000 re-sampled data the subtidal zone ofthe GulfofElat (Weidneret
sets. Scale bar represents 0.01 substitutions per al., 1996). Anothercloserelationshipwastoclone
nucleotide position. OCS54, aplastid rRNA sequence from anatural
phytoplanktonpopulationcollectedinthePacific
a bacterial community two orders ofmagnitude Ocean, off the mouth of Yaquina Bay, Oregon
greaterthan thatpresent in tissue ofVWF. (Rappe et al., 1998). The identified microalgae
with plastids clustering close to E30004315 fall
isoBliaotceshefmriocmalVcWhFaraacntderRi.saotdioonraobfilaellwcausltuisveafbulle MinicthreosgceonpeircaeOxdaomnitnealtliaoannodfSEk3e0le0t0o4n3e1m5ar(eFvieg.al7e)d.
in clustering each of these assemblages into morphology consistent with identification as a
distinctphenons. Insome cases, the presumptive
microalga in the eukaryotic phytoplanton, with
identity of isolates could be deduced by cigar-shaped cells about lOum long.
comparisonwithtypecultureswhichclusteredin
the same phenon. However, this approach must Strain NW002 clearly belongs to the genus
be used with caution because ofthe difficulty in Pseudoalteromonas, a genus with many marine
identifying marine isolates based on criteria representatives, on the basis of the close
generally established for readily-culturable phylogenetic relationship between this isolate
isolatesofmedicalsignificance. Inaddition,many and sequences ofstrains classified in the genus
isolates scorednegativeagainstalmostthe entire Pseudoalterowonas. The 16S rRNA sequence
biochemicalprofile,asisfrequentlythecasewith mostcloselyrelatedtothatofNW002wasderived
marineenvironmental isolates. Interestingly, two from a clone derived from a microbial mat at a
phenons from each sponge comprised hydrothermal vent site, the Loihi Seamount,