Table Of Content'
MOLECULARTECHNIQUES REVEAL WIDE PHYLETIC DIVERSITY OF
HETEROTROPHIC MICROBES ASSOCIATED WITH DISCODERMIA SPP.
(PORIFERA: DEMOSPONGIAE)
JOSE V. LOPEZ, PETERJ. McCARTHY, KATHLEEN E. JANDA, ROBIN WILLOUGHBY AND
SHIRLEYA. POMPONI
Lopez,J.V., McCarthy, P.J.,Janda, K.E., Willoughby, R.&Pomponi, S.A. 19990630: Mo-
leculartechniquesreveal widephyletic diversityofheterotrophic microbesassociatedwith
Discodermia spp. (Porifera:Demospongiae). Memoirs ofthe Queensland Museum 44:
329-341. Brisbane. ISSN 0079-8835.
Sponges are well known to harbor large numbers ofheterotrophic microbes within their
mesohyl. Studies to determine the diversity of these associated microbes have been
attempted foronly afewshallowwaterspecies. We cultured various microorganisms from
several species ofDiscodermia collected from deep water using the 'Johnson-Sea-Link
manned submersibles, and characterised them by standard microbiological identification
methods. Characterisation of a small proportion (ca. 10%) of the total and potential
eubacterial isolate collection with molecular systematica techniques revealed a wide
diversityofmicrobes.Phylogeneticanalysesof32smallsubunit(SSU) 16S-likerRNAgene
sequences from different microbes indicated high levels oftaxonomic diversity associated
—withthisgenusofsponge. Forexample, bacteriafrom—at least fiveeubacterial subdivisions
gamma,alpha,beta, CytophagaandGram positive wereisolatedfromthemesohylof
Discodermia. Several strains were unidentifiable from current sequence databases. No
overlapwas foundbetween sequencesof24 isolates and 8 sequencesobtained by PCRand
cloningdirectly fromspongesamples. Theabundanceanddiversityofmicrobesassociated
with sponges such as Discodermia suggest that they may play important roles in marine
microbial ecology, dispersal and evolution. O Porifera, Discodermia. microbialdiversity;
bacterialsymhionts, invitroculture, genesequencing, 16SrRNA.
Jose V. Lopez (email: [email protected]), Peter J. McCarthy, Kathleen E. Janda, Robin
Willoughby&ShirleyA.Pomponi,DivisionofBiomedicalMarineResearch, HarborBranch
OceanographicInstitution,5600US1North,FortPierceFL34946, USA; 16February1999.
Many marine sponges harbor numerous constituentorganismscoexistingwiththesesponges,
microbial symbiontsor'associates'. Researchers e.g. speciesrichness, ecological function, metabolic
enumeratingandcharacterisingsponge-microbial load,etc.Lithistidsingeneral, andDiscodermia in
interactions have shown that bacterial biomass particular, are the source ofseveral compounds
canreachover50%insomemarinespongeswith whh pharmaceutical potential (Longley et al.,
(cVoarcreelsepton&dinDgonpahdeanyo,me1n9a7l7l;yShimupgseond,ive1r9s8i4t;y a1l9.,911;99K4e)i.lIyd-eBnotrigfiecsateitonal.o,f1m9i9c4r;obGiuanlaasseskoeciraateest
Wilkinson, 1987; Santavy et al., 1985; Fuerst et jn Discodermia could lead to a belter under-
al., 1999,tinsvolume).Althoughstudiesotsponge- standingoftheecologicalorphysiologicalroleof
bacterial associations are hampered by upper these compounds. In this study, we provide a
mbeicbreoloorwga1n-i1s0m%c(uAlutsutriabnih1t9y88l,imiEtgsu,cehsit&imaItsehdidat,o pdirveelrismitiynaasrsyocdieastcerdiwpittihonspoecfieesuboafctDeirsicalodesrpemciiae,s
1990; Button et al. 1993), focus on marine using both microbiological and molecular tools,
Famioncrdrtoehbxeiaiamrlphleaecb,oilntooagvtyeslaaranesdpbeetcmatgxsrooenfvoedmaiylveeirdssbeiynmmcioreclaresocibdnegs. Aiflca•tionandma,tcrhfitn&hgiosfDe/x,ecr^dseemw/a,s7_atshseocidiaetnetd_
Mrg1c,e9ln9n8eTe-triRncesyHeiatlpvalpl,p^.re,oras1ic9n"ch9n5eoc;smD~im.s(tA)e.mle,atna,ln.,,e1itn9o8ao8l.;,VD*e1li9o9n5g,; umcunsiucicrunrrlgoe,tb.nietvt,hasebUes1lnqeepuMqoemluniye.ccmernesocrotear0sgtdeah,naeitc,csahlmbao,ssaiensswetesrrre.eaRcNPctnhA,ioaotrnr.eaencl(tat,,tPei.irCavi.Rles,)l,eiJ,yAdn
An understanding of the biology of marine cloning and DNA sequencing ofcloned 16S-like
sponges in the polyphyletic order 'Lithistida' small subunit(SSU)rRNAgenesegments(Paceet
would be enhanced by characterisation of the al., 1986; Lane et al., 1991; Delong, 1998).
)
330 MEMOIRS OF THE QUEENSLAND MUSEUM
Molecular phylogenetie analyses of these centrifugetubes at -75°C for30mins. About 1ml
sequenceshighlightthediversity oflineages found of sponge mesohyl (5-1Og ground in 20ml of
within a single sponge genus. Final systematic filtered sea water) was layered on top of the
resolution ofeach isolate basedonrRNAsequence thawed Percoll/CMF working solutions and
dataalone is not attempted in this study. centrifuged at 2,000 RPM for 20mins. Tubes
were punctured approximately 2.5cm from the
MATERIALS AND METHODS bottom, and five 2ml fractions were collected
into 24 well plates. About 100ul ofeach fraction
COLLECTION AND ISOLATION OF was then plated onto the appropriate isolation
MICROORGANISMS FROM MARINE media (see below).
MACROORGAN1SMS. Specimens belonging Isolation media used in this study were: 1
toDiscodermiaspp. usedas sourcesofmicrobial Chitin Sea Water: Colloidal chitin (in approx.
isolates characterised in this study are listed in 25ml deionised H-.O), 0.25g dry weight/liter
Table 1. Isolates from several non-Discodermia medium; agar, 20g; ASW, 975ml. 2) LN: Bacto
sponges (Dercitus, Halichondria and Corticium Peptone (Difco), 0.5s; Yeast Extract (Difco),
spp.) were included for a cursory comparison 0.5g; agar, 16g; 75% (v/v) ASW, 1L. 3) M3:
withmicrobeprofilesofDiscodermiaspp.(Table KTIP04, 0.466s; NaH,P04 0.732s; KNO, O.lg;
2). All specimens were obtained in accordance MgSO4.7H?0, O.lg; Na propionate, 0.2g; NaCl,
cwoiotphertahteinpgersmoivtesreaingdn rguolveesrngmreannttse.d Mbayritnhee 01.8209mg;g;CManCS003, 2200mmgg;;agFaerS,0148,g2i0n01mLg;deiZonnSis0e4d,
macroorganismswerecollectedeitherby SCUBA H2O.Cyclohex4i.mide 50mg)andthiamine(4mg)
(
or through the use of the 'Johnson- Sea-Link' were added after autoclaving. 4) ISP2: Difco
manned submersibles. On return to the surface, ISP2 prepared with 75% (v/v) ASW. 5) HSV
spongesamplesusedformicrobialisolationwere (humic acid, sodium salt), lg; glycerol phos-
immediately sub-sampled and prepared for phate, HOmg; 75% (v/v) ASW, 1L; 10ml BME
plating using the following method. The sponge Vitamin Mix (Sigma) added after autoclaving.
was surface-sterilised by rinsing with 70% (v/v)
ethanol. A cube ofmesohyl (approx. lem was Inoculated plates were incubated at ambient
) temperature (approx. 25°C) for2-4weeks. After
removed using aseptic technique and placed in
20ml sterile artificial sea water (ASW). The tthriasnspfeerriroeddtooffirnecsuhbpaltaitoens,odfitshcereitseolcatoiloonnimeesdiwuemr,e
sample was then ground in a Waring blender
pre-sterilised with 70% (v/v) ethanol, and the incubated and then re-streaked until the isolate
was axenic. Isolates were then transferred to
resulting suspension was serially diluted with
sterile ASW. The dilution series was used as the MarineAgar2216(Difco).All strainsusedinthis
studyweremaintainedasMarineAgar2216slant
inoculum for a series of isolation plates.
Typically, lOOulofthe 10°, I0"3and 10"5dilutions cultures.
were used since these were found to bracket the DNA ANALYSIS. Genomic DNA from bacterial
range at which discrete colonies were found. isolates and sponge mesohyl fractions was
Sponge cell dissociation and selective cell extracted using standard purification methods
enrichmentwereperformedbydissociating fresh (Sambrook et al., 1989), although some ofthis
spongesectionsincalcium-andmagnesium-free DNA required alternative purification methods
artificial seawater (CMF) (Pomponi & or modifications which have been previously
Willoughby, 1994) in a Virtis grinder for 1Osecs. described (Pitcher et al., 1989). Segments ofthe
Theresultingslurrywas filtered througha 70pm 6S-likesmall subunit SSU)nuclearrRNAgene
1 (
strainerandcellswerechecked forviability. Cell amplifiedfrombacterialculturesweresequenced
suspensionswerethenplacedinto 15mltribesand directly after purification of the PCR product.
centrifuged to obtain enriched fractions of Products derived from sponge mesohyl fractions
spongecells,unicellularbacteriaandfilamentous were cloned before sequencing based on the
bacteria. following procedure.
Several microbial isolates (e.g. K.200, K202, The universal eubacterial primers. Bcoli9 [5'
K261)wereobtainedbyfractionatingdissociated GAG TTT GAT CAT GGC TCAG 3'] and
sponge mesohyl through Percoll/CMF Gradients. Loop27rc [5' GAC TAC CAG GGT ATC TAA
A 10ml working solution of 15% Percoll/CMF TC3*],amplifyabout800bpofthe5' endofSSU
was diluted with 5M Tris [pH 8.0] from a 90% rRNA gene under standard PCR conditions
Percoll/CMF stock solution, and placed in 15ml (Lane, 1991). Thesegmentsencompass variable
HETEROTROPHIC MICROBES INDISCODERMIA 31
TABLE 1. ProfileofDiscodermiasamples used formicrobial characterisation. Key: 1,samplewasobtainedata
similarlocationanddepthasforthesamplesofspeciesnotbelongingtoDiscodermia{asindicatedinTable2).
2,Discodermiasample20-XI-97-1-001 islistedtwicebecauseitwasusedtobotha)isolate51 microorganisms
ofwhichtwohavebeenanalysed,andb)toderivetheeight PCR-ampIifiedproductscomprisingthe28 and29
clone series, whichwere notcultured.
Discodermiasponge Totalno.microbial
sampleID Locationcollected Depth(ft.) isolates No.sequencesanalysed
21-III-87-3-014 Bahamas 100 5 2
18-HI-87-3-001 Bahamas 515 2 7
8-XI-90-1-001 Bahamas 592 9
l-XII-92-2-001 Bahamas 110 13 1
7-XI1-92-2-00I ' Bahamas 540 8 1
17-XTI-92-1-005 Bahamas 535 10 1
15-1-96-2-012 Bahamas 520 1 1
27-X-96-1-003 Bahamas 543 59 7
29-X-96-4-006 Bahamas 520 8 1
9-XI-97-3-008 Ilonduras 383 65 i
I6-X1-97-1-004 Honduras 440 100 ?
20-XI-97-1-001 2 Honduras 415 51 2
20-XI-97-1-001 : 1londuras 415 8
rotals 331 32
regions V1-V4 ofbacterial SSU rRNA (E. coli conserved rRNA sequences relative to
positions 9-804) and were expected to provide Discodermia- derived microbes were identified
sufficient genetic variation for phylogenetic using queries generated by SIMILARITY-
analyses (Lane, 1991; Liesacket al., 1991). This RANK in the Ribosomal Database Project
size also facilitated complete sequencing ofboth (Maidak et al., 1994), or by BLAST using
strands with a minimal number of sequencing GenBank (Altschul et al., 1990). Preliminary
reactions, using the above amplification primers aliunments of sequences were made using
andinternalprimersint-250f[5' GAC TCCTAC P1LEUP (GCG, 1994), followed by a manual
GGGAGGCAG3']andint-275rc [5'CACGCG verification for the presence ofcanonical rRNA
GCG TCG CTG CAT 3']. The typical PCR secondarv structures and compensatory base
amplification profile was 94°C denaturation for changes (Neefs et al., 1993; Gutell et al. 1994)
45secs, 53-55°C annealing for60secs, and 72°C (also see Fig. 1). A gap extension penaltsy of 1,
extensionfor60secs,repeatedfor30cycles.PCR ratherthanthedefaultof4,maximisedsimilarity
products conforming to expected molecular by allowing longer gaps. Maximum parsimony
weights were purifiedby gel isolation orQiagen analysiswasperformedwithPAUP,version3.1.1,
columns (Qiagen), and sequenced by the dye- while neighbour-joining (NJ) and maximum
terminator cycle sequencing method (Applied likelihood (ML or DNAML) analyses were
BiosystemsInc-AB1)runonABI373 automated performed with PHYLIP 3.572 software,
DNA sequencers (University ofFlorida, 1CBR,
DNA SequencingCore Lab, Gainesville FL). To (Felsenstein, 1993; Swofford, 1993; Hillis et al.,
identify potentially uncultivable microbes, PCR p19e9r6f)o.rBmoeodtstarampirneiplmiucamtioonfs o1f00dattaismetesswaenrde
products derived from either total mesohyl
individual taxa (or operational taxonomic units,
preparations or enriched mesohyl fractions were
'shotgun' cloned into TA vectors according to o(rexOhiTbUist)ingwhliocnhgabppreaanrcehd lteonbgtehsproobrlemmaatniyc
manufacturers' instructions (Invitrogen). Clones
uninformative nucleotide substitutions) were
derivedinthismannerweredesignatedanumber
jackknifed (Efron, 1982). Typical heuristic
(bteogtialnnmiensgowhiytlhperietphaerra2t8io(ne)n.riched fraction)or29 searches in PAUP utilised evaluations ofat least
50 replications of random sequence additions,
All rRNA gene sequences were analysed with tree bisection-reconnection (TBR), and decay
the GCG DNA analysis package (GCG, 1994) indexassessments(Hillisetal., 1996). Subsetsof
and SEQED data editor (ABI). The most the total dataset of over 40 OTU's (including
..
332 MEMOIRS OF THE QUEENSLAND MUSEUM
stem loop stem
13a 13 13a'
B853 CCAAGGCGACGATTCCTAGCTGGT-CT GAGA GGATGAT-CAGCCACACTG
K121 CTA - -
M485 C.T CTA -T. T -. . . .A
H529 C G.G C.AC- G. . . .-.G
K146 CGGG C..C- GC. .C-.G
K275 C.A - -
29B2 .,T. ..CGA. . ..A.GC- G.
29A G.G. C.AC- ..G -.G
28C A. .C.C.T.. . .C. .G.G CGC.Y.
B
stern bulge stem loop stem bulge stem
17a 17a 17b 17 17b' 17a' 17a'
B853 TGATGCAGCCATG-CCGCGTGTATGAAGAA GGCC TTC-G GGTT GTAAA--GTACTTTC
J131 - G - --.C
K127 - G - —~.C
E034 -..C G - .C
B849 C A...- GC - -—-.£ T
K121 ....C - G...T..C A- —.C
H485 ....C - AG. . .T A-. ...C —.CT
H529 . . .C.G. ..A.C.- AG. . .T TT ...-. .A.C ACT. .G.T
29B2 ...C A.C.C G.G..C TT ..TC. .A C—TTCC. . .AA
28D2 C C- GC..T - —-C T
29A C - AG...T..C A- .CT
FIG. 1. Representativepartial alignmentsofSSU rRNA regions with conserved secondary structures (Neefset
al., 1993; Gutell etal., 1994). Structurescorrespondingto: A, Stem/Loop 13; B, Stem/Loopand Bulge 17are
shown over the nucleotide sequences. Both strands ofa stem are underlined with the downstream structure
marked by a (k) above the sequences. Stem 13 in (A) and stem 17 (B) begin at E. coliposition 288 and 405,
respectively. Stem 13a' contains an asymmetric bulge, lengthening it relative to upstream 13a. Nucleotides
identical to the first reference sequence are shown below as dots (.); gaps are indicated by (-); compensatory
mutations in all stems are underlined.
outgroups listed below) were analysed to verify andincludedinphylogeneticanalysesdespitethe
support foreach clade. ForNJ, genetic distances shorter length ofrRNA sequence.
were calculated with DNADIST, also in Sequences(withGenBankaccessionnumbers)
PHYLIP, using Kimura's 2N parameter correct- ofthe followingrepresentative eubaclerial strains
ion for multiple substitutions and empirically and genera were used as outgroups or as ref-
derived transition/transversion ratio of 2.0. erence sequences for the major clades observed
Althougheachmaximumlikelihoodanalysiswas in phylogenetic reconstructions: Bacillusfirmus
limited to a subset of 20 OTUs, different taxa (X60616), Pseitdomonas straminea (D84023),
within major clades were interchanged and Capnocytophaga sp. (X97245), Aiteromonas
substituted in separate runs of the program to macleodii (X82145), Vibrio alginolyticus
monitor consistency ofthe consensus topology. (X74690), Ridgeia piscesae (U77480),
NJ and DNAML analyses were performed on a Thermotogamaritima(M21774),Actinomycetes
Digital AlphaServer 8400 mainframe computer spp. (X92705), Rhodobium marinum (M27534),
maintained at the Frederick Biomedical Super- Burkholderia solanacearitm (U28232),
computer Center in Frederick, Maryland. Clostridiumsp. (L09175,X77837), Chloroflexm
(D38365), Lyngbya (AJ000714) and an
Chimeric sequences were detected by com- unidentifiedmarinobacter(U61848).
paring the identity of 5' and 3s ends separately
beforemakingcontiguousconstructs,orbyusing RESULTS
theCHECK-CHIMERAoptioninRDP(Liesack
et al., 1991; Maidak et al., 1994; Rheims et al., MICROBIAL ISOLATIONS. Phenotypic
1996).Whenchimericproductswerefound,each identifications ofmicrobial isolates were made
respective terminal sequence was analysed as a by analyses ofcolony morphology, microscopic
singleentityuptotheartificialcrossoverjunction observation and Gram staining. A general
HETEROTROPHIC MICROBES IN D1SCODERM1A
TABLE 2. Comparison of isolates from different when sequence identity' exceeded 99% (Fox et
sponge species collected at similardepths, locations al., 1992). Indeed,itisprobablymoreappropriate
and times as for samples ofDiscodermia (listed in to make reference to a specific 'rRNA type' or
Table 1). The number given for Discodermia is the strain than to infer that these identifications are
average forthe three samples analysed.
homologousto species-leveltaxonomy. 2) Since
No.ofIsolates it was not possible to obtain multiple operon
Spongetaxon Nsofao.mrpoaltneasslpyuossniegsde pGosriatmi-ve nGegraatmi-ve Fungi tsheeqrueefnocreesnoortspaomspsliebsleoftoanayssgeisvsenpoissosliabtlee, iitnwtraas-
bacteria bacteria strain or intraspeeific variation amongst the
Discodermia 3 5 3 1.5 microbial taxa (Clayton et ah, 1995).
Dercitus 1 4 4 The widetaxonomic diversity observed in this
Carticium 1 8 1 survey was striking. For example, at least three
Halichondria 1 3 1 3 major eubacterial divisions, or five classes
(Woese, 1987; Balows et al., 1992), are
taxonomic grouping of microbial isolates was represented in the microbial isolates obtained
from samples of Discodermia: the gamma-,
obtained from differentsponge taxaproximal to,
beta-, alpha- proteobacteria, cytophaga, and
or at similar location, depth and time as
Discodermia samples (Tables 2, 3). Although Gram-positive eubacteria. Of the 24 isolate
major microbial groups such as eubacteria, sequences obtained from Discodermia the most
commonly observed bacterial subdivisions were
actinomyectes andfungi were identified, archae- gamma-proteobacteria (9) and aipha-
bacteria and protists were not cultured. Fungal
isolatesandtheirsequenceswillnotbediscussed proteobacteria (8), followed by Gram -positive
in this paper. To investigate any possible trends (4), beta-proteobacteria (2) and possiblv asingle
or biases in microbial isolations, the number of Cytophaga-like isolate (K279) (Table'4). This
isolates in different microbial categories Gram-negative isolate matched most closely a
psychrophilic marine Cytophaga, with some
obtainedfrom differentspongetaxaanddifferent
subsampies are summarised in Table 3. These regionsreaching93%correspondenceinidentity,
values indicate thatthere are differentprofiles of although these sequences were still not fully
microbialpopulationsamongdifferentspeciesof confirmed at the time of writing. Nonetheless,
detection of Cytophaga primarily from the
sponges, and thatthere is also potential variation
inmicrobial yields among different samples ofa surfaces ofmarine aggregate particles in marine
systems has been previously described (Delon»,
particular sponge species. For example, some
1998).
variation in isolate yields appeared to be related
togeographical differences in sample collection, There were only a few cases where sequence
whereas specimens of particular species from database matches appeared unequivocal: e.g.
differentdepthsdidnotexhibitanystrongtrends. K169 showed high similarity to Pseudomonas
Somevariationmayalsobeattributedtochanges stutzeri, a common Gram-negative microbe in
in thecriteriausedto selectcolonies forisolation RNA group 1 (Balows el al., 1992), and E034
and in the mediaused for certain experiments. appeared to be related to a bacterium first
characterised from Pele's hydrothermal vents in
EUBACTERIAL STRAIN IDENTIFICATION. the Pacificocean.InotherinstancesDiscodermia
The names ofeubacterial strains resulting from isolates matched sequence database entries at
highest identity scores are listed in Table 4, highly significant identity levels (97-100%
together with their corresponding percentage similarity), but these matches were made to
sequence identities. Relatively good agreement 'anonymous' strains identified only to the genus
was observed between the two major sequence or even subdivision level. For example, the
datasources, RDF and GenBank databases, with closest relatives to isolates K171 and J131 were
novel bacterial rRNA sequences from cultured an"alpha-proteobacterium MBIC3368'undAltero-
isolates used to infer possible taxonomic monas sp., respectively. Moreover, comparison
placements. However, several caveats to this ofthese two isolates maintained high sequence
procedure should be emphasised. I) Database conservation acrossthewholerRNA segment, in
searchesrevealedonlythemost similarsequence contrast to 28D which had a range ofconserv-
in the respective database, and these were not ation values (77-93%) largely dependent on the
considered as an absolute identification of a region ofthe gene under comparison (Table 4).
particular 'species' or strain of bacteria, even Since several species of Vibrio exhibited equally
334 MEMOIRS OF THE QUEENSLAND MUSEUM
TABLE 3. Profile of isolates from different sponge species collected at depths and habitats similar to
Discodermia. Bold face type indicates samples that were obtained at similar locations or on the same
expedition, andthus reliable formoredirectcomparisons betweensamples.
Numberof Bacteria
Spongetaxon sfaomrpilseoslautisoend Depth(ft.) Collolceactitoinon GramPos. GramNeg. Actinomycetes Fungi
Halichondria 21 390 W. Barbados 1
526 CanaryIslands 1
477 Bahamas 2 4
393 Bahamas 4 1 1 2
1386 Bahamas 5 1
750 Bahamas 1 1 1 1
1803 Bahamas 5 1 1
803 Bahamas 2 1 1
440 Bahamas 1 1 1
576 Bahamas 4
543 Bahamas 3 1 3
473 Bahamas 1
479 Jamaica 4 9 1
520 Jamaica 3 2
1497 Jamaica I
410 Jamaica !
480 Bahamas 6 3
230 Bahamas 1
149 Florida,eastcoast 12 33 3 5
472 PuertoRico 3 73 2
450 PuertoRico 1
Derailus 10 20 Venezuela 2 3
455 Bahamas 1 4
504 Bahamas
540 Bahamas 1 1
432 Bahamas 6 1 1
700 Bahamas 6 3 1
525 Bahamas 4 4
806 Jamaica 3 1
400 Jamaica 15 1 3
384 USVirginIslands 3
Corticium 9 581 Bahamas 1
525 Bahamas 8 1
462 Bahamas 1 2 1
443 Bahamas 4 8 1
305 Bahamas 2
2819 Bahamas 1 3 1
377 Bahamas 8 1
1585 Turks&Caicos 1
90 PuertoRico 5 11 2 3
Discodermia 10 592 Bahamas 1
110 Bahamas 7 5 1
540 Bahamas 4 3 1
535 Bahamas 3 2 5
520 Bahamas 8 5 1
543 Bahamas 21 31 5 2
520 Bahamas 3 2 3
383 Honduras 9 51 1 4
440 Honduras 36 61 1 2
415 Honduras 14 25 12
HETEROTROPHIC MICROBES IN DISCODERM/A 35
highscoresincomparisontotheisolateK261 (ca. Flectob%acilius, along with some conservation
98%), only the genus is listed in Table 4. (82-91 )toRidgeaandRiftlahydrothermal \ent
Although we did not deliberately attempt to bacteria (Feldman et al., 1997). Sequences o\'
other Discodermia isolates exhibited significant
detect or amplify cyanobacteria, this group is similarity to bacteriaassociatedwithhydrothermal
well knownamongst sponges livinginthephotic vent habitats and organisms. Also, clone 29B2
zone (Wilkinson, 1987; Ruetzle^ 1990; Diaz,
appeared to be distantly related to Clostridia.
1997), and some ofour samples ofDiscodermia Furthermore, many microbes have not yet been
were collected in or near this zone. Clone 28C analysed or may nothave been isolated from the
exhibited strong sequence similarity to a
original plates. Interestingly, the sequences of
LeucothriX) which has been described as a large- most ofthe PCR-derived rRNA clones exhibited
dgliiadmientgebra,ctemroiraprheollatoegditcoaclylaynodbiascttienrcita,(mBaarlionwse irdeecntoimtmyelnedveeldstbheatlouwnid9e0n%t,ifiaenddsttrhauisnsitwimtahythbies
etal., 1992).
level ofconservation to any entry in either data-
To facilitate direct comparisons between our base be considered strong candidates for 'novel1
sample isolates and known sequences, and to species designation.
monitorsamplingvariation,weundertookparallel
rRNA sequence analysis ofpotentially different PHYLOGENY OF NOVEL ISOLATES.
microbial isolates from other sponge taxa living Phylogenies constructed from the rRNA
ingeographicalproximitytoDiscodermia(Table sequenceswereusedtocharacterisethediversity
2). These isolates (indicated inboldface in Table and relatedness ofDiscodermia bacterial strains.
3) were derived from sponge samples collected Figures2 and3 displaydendrogramsconstructed
from the same habitats and depths as Disco- withtwodifferentphylogeneticalgorithms:maxi-
ddaetram,iaalt(ihnoduigchatperdeliinmbinoalrdy,insTuagbgleest1a).hTihgehseerffreew- rmeuspmectpiavrelsyi.moMnyaxainmdumneilgihkbeoluirh-ojoodin(iMngL)(NaJn)-,
quency of a Bacillus in non- Discodermia alyses were also performed with smaller subsets
sponges, which is possible circumstantial of taxa. Relationships constructed under the
evidence for sponge-specific microbial assoc- principal ofparsimony use a criterion of mini-
iations (Althoffet al., 1997) (see Table 3). More mum evolution (shortest tree), while NJ uses a
extensive comparisons could be designed to clustering algorithm based on overall similarity
determine the optimal and natural conditions of or distance in a comprehensive OTUxOTU
Discodermia-Bssociated microbes, perhaps by matrix ofcorrected pairwise distances (Hillis et
wider sampling of proximal sponge and non- al., 1996). Although NJ and ML reconstructions
sponge habitats (e.g. sediments, seawater, etc.). better compensate for rate variation among
different lineages(Hasegawa& Fujiwara, 1993),
PCR was also used to directly amplify rRNA ML analyses involved fewer taxa due to com-
sequencesfroma)atotalspongecellpreparation, putational limitations forlarge datasets, and thus
and b) a dissociated mesohyl fraction enriched are not discussed further in this work.
AfolrthsopuegchifoinclypoapuslmaatlilonnsumobfermiocfrroRorNgAancilsomnse.s Despite relatively large differences in rRNA
sequences among some of the taxa, sequence
wereobtainedbyshotguncloningfromthesetwo
sources(labelled28A-Zfortheenrichedfraction alignments appeared to be robust. Multiple
invariant positions and highly conserved regions
and 29A-Z for the total 'crude' mesohyl prep-
correspondingtopreviouslydescribedsecondary
aration),theiridentitiesandoverallcompositions
structures (e.g. loop 20. loop 14-15, and stem 5)
appearedto be different fromthose derived from
were observed by eye along the nearly 800bp of
culturedisolates(Table4).Inspiteofthefactthat rRNA sequences. Observation ofcompensatory
these sequences also exhibited the highest
afflrr.,aegq1mu9ee9nn1tc),y, psorfoemccehliumodeifrnitgchaePnsaeClyRcslioasrnteoisffatcmhtaesyt(oLtriaeelpsrraecRskNeneAtt cminountstaheterivoanntosivioennlstobefamctthe1or3isaaelndstrbrRuuNlctgAuesre1s7c,o(arFrmigoo.bnogrI)a.otteOhenrtlshy,e
'uncultivable' microbes. Up to 99% ofnaturally onehighly variableregion corresponding to loop
occurring microbes may be overlooked by 11 (Neefs et al., 1993; Gutell et al., 1994) re-
quired removal due to ambiguous alignment and
Astmaandnanrdectuallt.u,ri1n9g95t;ecHhunligqueensho(tBuzt&tonPaetcea,l.,11999963);. its effect on nucleotide site homology.
For example the 5* ends oftwo clones in the 29 For the 30 microbial taxa analysed by max-
series appeared to significantly match imum parsimony (Fig. 2), an estimation of
336 MEMOIRS OF THE QUEENSLAND MUSEUM
TABLE 4. Discodermia-associated microbes identified by 16S-like rRNA sequences. Key: 1, the list of
organismswasderiveddirectlyfromtheoutputofGenBankorRDPqueries(Altshchuletal., 1990; Maidaket
al., 1994). Thenamesoftheclosest relativemayreferto species, genusorcommonnames. 2, percentidentity
wasderivedfrom BLASTscoresonly,and mayreflectidentitiesofdifferentsegmentsofasinglequeryrRNA
sequence. Thus, variable conservationofdifferentregions ofthe rRNA sequence is indicated intheranges of
identity values shown. 3, sincethese cloneswere showntobe chimeric, wholecontigs were notanalysed and
query results reflect identities for5' termini only. 4, five microbial sequences derived from now-Discodermia
sponge microbes are underlined - M234 and M196 (Corticium), M162 and M099 (Halichondria) and M119
(Dercitus).5,clonalsequencesindicatedwithanasterisk(*)representthe28/29clonalserieswhichwasderived
from PCR amplification of sponge mesohyl fractions. Therefore, these sequences were not derived from
cultured isolates.
Mostcloselyrelatedgenus MicrobialID %Sequence Mostcloselyrelatedgenusor MicrobialID %Sequence
orgroup no. identity" group1 no. idientity"2
ALPHA BETA
Alphaproteobacteria K200 90 Unidentifiedmarine K255 92-96
proteobacterium
AlphaproteobacteriaMBIC3368 K202 97
Alcaligenes 28D2* 85-94
Alphaproteobacteria K.121 88
Betaproteobacteria B849 92-99
Alphaproteobacteria KI26 91
GRAM-positive
Alphaproteobacteria K275 88-96
Bacillus(lowGC) M680 96
Alphaproteobacteria M485 94
Bacillus(lowGC) M529 97
Alphaproteobacteria MI624 97
Bacillus llrmus M0994 91
Erythrobacter E035 98
Bacillusfusiformes 2SX* 81
AlphaproteobacteriaMBIC3368 K171 99
Bacilllusfirmus M1964 97
Phodospirillum 29A* 90
GAMMA |Bacillusfirmus M2344 91
Unknownactinomycete 28D* 91
Hydrothermalventbacterium M1I94 97 1
Nocardia,actinomycete K146 97
Vibrio B853 96
Nocardia,actinomycete K145 80
Alteromonas J131 100
CYTOPHAGA
Hydrothermalventbacterium E034 99
Marinepsychrophile K279 94
Vibrio K261 98
AMBIGUOUSGROUPING
Pseudoaltermonas K127 97
Flectobacillus 29W* 85
Vibrioalginolyticus K141 97
Flectobacillus 29B3 82-91
Microbulbifer C724 89
Clostridia 29B2* 76-81
Unidentifiedgamma C723 91
Pseudomonasstutzeri K169 99
Leucothrixmucor 28C3* 80-94
skewness of tree length distributions (i.e. for supportislikelyWduetotheuncertainplacementof
10,000 random trees using the Random Trees clones29B, 29 and28C. ProximalRidgeiaand
option in PAUP), yielded a gi statistic of-0.64. Marinobacter groups are typically grouped with
Thisvalueisabovethe99%significancelevelfor gammaproteobacteria.
the corresponding critical value of gi for more Inparallel,neighbour-joininganalysesofrRNA
than 25 taxa, indicating a strong leftward skew-
sequencedatayieldedverysimilarconclusionsto
ness andhigh phylogenet&ic signal in a four-state parsimony(Fig. 3). Pairwisegenetic distances of
character dataset (Hillis Huelsenbeck, 1992). all OTU's based on Kimura's 2N parameter
Weighting transversions over transitions byMPa correction (Hillis et al., 1996), ranged from
factor of 2 shortened the overall length of 0.04->0.70. The major differences between the
trees. There were only two and four more trees NJ and MP trees were: 1) higher bootstrap sup-
that were one or two steps longer, respectively, portforindividual cladeswithNJrelativetoMP;
thantheshortesttreeshowninFigure2usingthe 2) fewer collapsed nodes and polytomies with
same dataset. The clade containing beta and NJ,providingclearergroupingsofmajorproteo-
gamma bacteria shows the weakest support bacteriasubdivisions;3)inclusionofRhodobium
(55%), and is thus depicted as apolytomy. Low andclone29AwiththeclusterofAlphaeubacteria;
HETEROTROPHIC MICROBES IN DISCODERMIA 337
inn K2B68I53 status ofeach novel isolate, some ofthese taxo-
JI3I nomic placements are likely to be revised in the
KHJ E034 future.
W-MI19
67 tcromonas Nevertheless, the topologies ofparsimony and
K169
B849 distance trees were generally consistent in
Burkholderia ]" showing at least 5 major clades. Isolates K261
55 «100290*28D2- and K171 wereatthetipsofthe MPtreeandthus
29IV* their omission to accelerate computation times
2SC* did not have a significant effect on NJ tree
^~ MRairdigneobiaactcr topology. Several features in the present recon-
i—— Mivi4-85 structions ofDiscodermia microbes, such as the
KK227050 a monophyly ofall proteobacteria, strongbootstrap
1—00' KI71 ] supportforthegammaandbetasubdivisions,and
29A'
K279 ]c the outgroup status ofthe Gram-positive clade,
i P
SS rM52R9hoLdOoObiuniCytophaga Maroeralelisnpaegcirfeiecmaellnyt,wiathlocunrgrebnrtabnaccthericahlartaacxtoenroimsye.d
' Bacillus the lineage of K279 and its strong association
K12486D* IGr+am with Cytophaga bacteria. This branch is con-
891 Actinomycetes sideredbyustobeafifthmajorbacterial cladeof
—29B2* the sponge. Relatively long branch lengths were
Chloroflexus
• prominent for several other lineages (e.g. K146
FIG. 2. Representative maximum parsimony and some clones in the 28/29 series).
phylogeny of Discodermia-associated microbes. Although uncultivated clones 28C, 29B and
Cultured isolates are shown in standard bold font. 29W were grouped significantly with other
PCR-derived/uncultivated clones are shown with marine bacteria discovered from previous en-
amsitcerroisbkesM(*i)la9ndisiunndietarlliicns,edw.hiNluembneonr-sDibsecluodweremaicah vFiurhornmmaennt&alDavsiusr,ve19y9s7),(Mthoeydeartaebtasealm.,at1c9h9i5n;g
nReopdreesreefnetrattiovtehseobfogoetsnterraapovraflaumeilaifteesra5re00liistteerdatiinotnhse. of 29W and 29B to Flectobacillus (a Cyto-
Methods and have names written out. Preliminary phagales) indicates that accurate placement of
heuristicsearches usingthesamedataset, 10random these bacteria require more refined determin-
stepwise additions of taxa with tree bisection ation. However, the tight clustering of taxa
reconnection (TBR), found only 2 most parsimon- observed also suggests possible endemism or
ious trees. Length of the two most parsimonious ecological specificitywithrespecttoDiscodermia.
heuristic trees was 1864 steps, with a consistency Consistent with the database matches to Clos-
index (CI) of 0.495. Sub-optimal trees that were tridia, clone 29B2 was placed repeatedly near
pleocntgievrelbyy,a1ndorre2tasitneepdstnhuemsbaemreebdasoincltyo2poalnodgy4,ofrtehse- outgroup taxa at the base ofall trees. The weak
bootstrapconsensus. Morever,theheuristictreesdid and unresolved positions ofsome taxa, such as
notcollapsethebetaandgammaproteobacteriaclades 29B2, 29A and K146, connote another level of
into a single polytomy, but rather showed the beta diversity. The inclusion of non-Discodermia
proteobacteriaasadistinctgrouprelativetothegamma isolates(M119.M169,M234,M162orM099)in
bacteria(McDonaldetal., 1997). Preliminarygroup- some reconstructions did not significantly alter
ingsintheproteobacteriasubdivisionsandCytophaga treetopologies,nordiditsuggestanyevidenceof
(CP) were based on the identities obtained from taxon-specificsymbiosesoccurringinthecurrent
BLAST sequence database searches.
dataset.Overall,theseresultsparallel earlierdes-
criptions (Santavy et al., 1990) describing major
and 4) monophyly of all representative Gram- bacterial groups such as Vibrio, Aeromonas, and
positive bacteria. Phylogenetic assignment of coryneform/actinomycete (Gram-positive)
clone 29A, which appeared most closely related strains derived from marine sponges.
to the alpha subgroup, was problematic with all
threealgorithms(MLtreenotshown).Thedecay DISCUSSION
index for any group that included 29A was
always low (<3). Since it is not within the scope Phenotypic, comparative DNA sequence and
ofthis study to evaluate the strengths and weak- molecular phylogenetic analyses confirmed the
nesses of various phylogenetic methods, or to presence ofatleastfivedistincteubacterial clades
make definitive conclusions on the taxonomic of 6S-likeSSUrRNAsequencesfrommicrobial
1
)
338 MEMOIRS OF THE QUEENSLAND MUSEUM
Bacillus sequenced at the time of writing. Nevertheless,
M529 our finding that many different bacterial strains
K146 Gram and rRNA 'types' stem from only one sponge
ActinomvctUs +
28D* genusisnovelanddistinguishesthepresentstudy
Ml19 from earlierresults.
B034
Bacterial symbionts occur both intracellularly
J131
Alteromonas and extracellularly with respect to their sponge
B853 host mesohyl (Vacelet & Donaday, 1977; Simp-
Klf,<> 29W* 7 son, 1984; Wilkinson, 1987). However, without
sac* positive identification ofthe types ofinteracting
90" 29B* organisms, elucidating symbiotic parameters
Marinobacter such as nutrient transfer, detoxification or gene
Ridgeia exchangewillnotbeasmeaningfulasthosemade
B849
DO"-~ Burkholderia for well-established cnidarian-dinoflagellate
2A8*02* associations (Trench, 1993). It is possible that
29 some ofthe microbes identified here stem for-
M485
K200 (X tuitously from the microbial pool derived from
96 K275 sponge filter-feeding activities (Reiswig, 1971;
Rhodobium Pile et al., 1996). A long-standing question in
Cytophaga ]Cp sponge-microbial symbioseshasbeenhowdoso
100 K279
29B2* many different symbionts coexistand seemingly
Chloroflexus thrive in the relatively inhospitable (phagoctye-
filled)environmentofthemarinespongemesohyl
FItGhe.N3e.iDgihsbtoaunrc-ej-obiansiendgmpehtyhloodg.enTyherescaomnesttrauxcate(dexwcietpht (Simpson, 1984; Wilkinson, 1987)? One answer
K.I71 and K261) and annotations as that shown in may stem from the advantages of 'ecto-
Fig. 2 were analysed. symbioses1 and bacterial communities (Bull &
Slater, 1982).
isolatesofthelithistidspongegenusDiscodermia. Conversely, it is not unreasonable to suppose
Several ofthe identified Discodermia bacterial thata fractionofthebacterialspeciesisolatedand
groups, such as the gamma proteobacteria, are characterised here represent bona fide obligate
consistent with previous characterisations of symbiontsofDiscodermia,anexpectationwhich
deep-seamicrobes (Moyeret al., 1995; Feldman hasbeenconfirmedinothersponges(Burlandoet
etal., 1997;Fuhrman&Davis, 1997),whileother al., 1988; Althoff et al., 1998). Although not
lineages (e.g. K279, 28C, 29W, 29A) appear trivial, the question of determining specific
unallied ornovel. This situationmayhave arisen microbial symbionts could be approached by
viaacceleratedsubstitutionrates,whilegroup-or probing for consistent rRNA (or other genetic)
strain-specific synapomorphieswere maintained signaturesamongamatrixofgeographicallysep-
(Hillis et al., 1996; Peek et al, 1998). More arated samples of Discodermia present in our
likely,however,nocloserelativesexistincurrent collection. Definitive conclusions onthe relative
prokaryotic rRNA sequence databases, which abundance of a particular bacterial strain in
Discodermia are precluded, since the quanti-
underscores possible missing links in current
bacterial rRNA taxonomies. Similar to many staetqiuveenrceicnogvemrayyofsusgegveesratlamniycroofbtihaelftoylpleoswidnugr:ing
earlier surveys of bacterial diversity from the 1
a dominant presence in the host sponge and
environment(Paceetal., 1986;Giovannonietal., concomitant functional role in Discodermia
P19C9R0;aAnmdanmnoleetcuall.,ar19m9e5;thRohdesimhsaveet alp.,ro1b9a9b6l)y, ephxypseiroilmoegnyt;al2)bihaasbiotaftP-sCpeRcipfricimdeirffbeirnendciensg;soirte3s),
revealed unique microorganisms which are genomic DNAquality, etc. (Rheimsetal., 1996).
otherwise uncultivable undertraditional methods. Itwould be interestingin the future to determine
Although some variation may be attributed to whetherthemodeofmolecularevolutioninthese
differences among species of Discodennia and microbes matches other observations of faster
betweenindividual samplesofparticularspecies, nucleotide substitution rates in symbiotic versus
this characterisation also likely underestimates free-living marine bacteria, which may be a
total diversity inthegenus, sinceonlyabout 10% function of small population sizes of some
of the Discodennia isolate collection has been symbiotic communities (Peek et al., 1998).
