characterisation of bacterial communities associated with ......triplicate cell counts using a...

Characterisation of bacterial communities associated with toxic andnon-toxic dino£agellates:

Alexandrium spp. and Scrippsiella trochoidea

Georgina L. Hold a;*, Elizabeth A. Smith a, Michael S. Rappe b;1,Elizabeth W. Maas c;2, Edward R.B. Moore d, Carsten Stroempl d, John R. Stephen e;3,

James I. Prosser e, T. Harry Birkbeck f , Susan Gallacher a

a Fisheries Research Services, Marine Laboratory, Victoria Road, Aberdeen AB11 9DB, UKb Station Biologique, Centre National de la Research Scienti¢que et Universite Pierre et Marie Curie, BP 74, 29682 Rosco¡ Cedex, France

c Department of Microbiology, University of Otago, P.O. Box 56, Dunedin, New Zealandd Division of Microbiology, National Research Centre for Biotechnology (GBF), 38124 Braunschweig, Germany

e Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UKf Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK

Received 27 February 2001; received in revised form 12 July 2001; accepted 13 July 2001

First published online 8 August 2001

Abstract

Several dinoflagellate species have been shown to produce potent neurotoxins known as paralytic shellfish toxins. Evidence is alsoaccumulating that marine bacteria associated with dinoflagellates play a role in the accumulation of paralytic shellfish toxins. In this study,the diversity of bacteria in cultures of both toxic and non-toxic dinoflagellates, Alexandrium spp. and Scrippsiella trochoidea, were comparedusing colony morphology, restriction fragment length polymorphisms, denaturing gradient gel electrophoresis of PCR-amplified 16S rRNAgenes and, ultimately, sequence determination of the 16S rRNA genes. The results suggest that a number of different bacterial species areassociated with dinoflagellates, some of which are common to each of the dinoflagellate cultures examined, whereas others appear to beunique to a particular dinoflagellate. The phylogenetic diversity of the bacteria observed was limited to two bacterial phyla, theProteobacteria and the Cytophaga-Flavobacter-Bacteroides (CFB). Although phylum level diversity was limited, many distinctphylogenetic clades were recovered, including members of both the K- and Q-subclasses of the Proteobacteria. Additionally, several ofthe bacterial phylotypes isolated were not closely related to any published bacterial species but, rather, were identical to isolatescharacterised from Alexandrium cultures 4 years earlier. Finally, many of the bacteria isolated from the dinoflagellate cultures were relatedto microorganisms with known surface-associated life histories (e.g. the CFB phylum, Hyphomonas, Caulobacter and some members of theRoseobacter clade including Ruegeria algicola). ß 2001 Federation of European Microbiological Societies. Published by Elsevier ScienceB.V. All rights reserved.

Keywords: Dino£agellate ; Marine bacterium; Paralytic shell¢sh toxin; Polymerase chain reaction-restriction fragment length polymorphism; Polymerasechain reaction-denaturing gradient gel electrophoresis

1. Introduction

Interactions between algae and bacteria are commonlyobserved in both freshwater and marine ecosystems withbacteria increasingly cited as potentially important regula-tors in processes of algal bloom initiation, maintenanceand decline [1]. Bacteria are an inherent part of the phys-ical environment of dino£agellates, both in vitro and invivo and, as such, may be considered dino£agellate sym-bionts [2,3]. However, the ecological signi¢cance of mostnaturally occurring bacterial^algal associations is unclear

0168-6496 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 1 6 8 - 6 4 9 6 ( 0 1 ) 0 0 1 5 7 - X

* Corresponding author. Present address: Rowett Research Institute,Greenburn Road, Bucksburn, Aberdeen AB21 9SB, UK. Tel. : +44 (1224)71 27 51; Fax: +44 (1224) 71 66 87.

E-mail address: [email protected] (G.L. Hold).

1 Present address: Department of Microbiology, 222 Nash Hall,Oregon State University, Corvallis, OR 97331, USA.

2 Present address: Institute of Environmental Science and ResearchLimited, P.O. Box 50-348, Porirua, New Zealand.

3 Present address: Crop and Weed Science, Horticulture ResearchInternational, Wellesbourne, Warwick CV35 9EF, UK.

FEMSEC 1273 9-10-01

FEMS Microbiology Ecology 37 (2001) 161^173

www.fems-microbiology.org

and, in most cases, the bacterial species involved have notbeen identi¢ed. This is particularly the case with algaeassociated with the occurrence of toxins in shell¢sh, suchas the paralytic shell¢sh toxin (PST) producing Alexan-drium spp.

Although the value of 16S rRNA gene sequence datafor identifying bacteria is rarely disputed, this informationis severely limited for bacteria associated with toxic marinedino£agellates. A recent study by Prokic et al. [4] inves-tigated the diversity of bacteria associated with Prorocen-trum lima, a known diarrhoetic shell¢sh poison producingdino£agellate, via microbial cultivation and 16S rRNAgene cloning and sequencing. The authors found Roseo-bacter-related microorganisms of the K-Proteobacteria todominate the P. lima micro£ora. Babinchak et al. [5] used£uorescently labelled 16S rRNA targeted oligonucleotideprobes to identify bacteria from toxic Alexandrium spp. asmembers of the K- and Q-Proteobacteria. However, resolu-tion in this study was low, and a more detailed character-isation would be required to resolve any di¡erences be-tween the bacterial populations of toxic and non-toxicdino£agellates. Several authors have also reported thatbacteria isolated from Alexandrium spp. cultures are capa-ble of producing PST but, again, limited information ontheir identity in the form of 16S rRNA gene sequence datais available [6^9].

The issue of bacterial PST toxigenesis is currentlynot resolved [3]. However, previous studies in our labora-tory have indicated that the bacteria associated with cul-tures of the dino£agellates Alexandrium lusitanicumNEPCC 253 and Alexandrium tamarense NEPCC 407in£uence the toxicity of these dino£agellates [10]. In orderto elucidate the contribution of particular bacterial speciesto dino£agellate/shell¢sh toxicity and bloom dynamics,it is essential that the ecology of bacteria associated withmarine dino£agellates such as Alexandrium spp. bestudied. As a prelude to investigating algal^bacterialdynamics in the environment, it is necessary to establishwhich bacterial taxa are involved, initially requiringhigh resolution characterisation of the bacteria associatedwith dino£agellate cultures. To this aim, using a com-bination of phenotypic (colony morphology) andmolecular-based techniques (16S rDNA restrictionfragment length polymorphisms (RFLP), denaturinggradient gel electrophoresis (DGGE) and, ultimately, se-quence determination of PCR-ampli¢ed 16S rRNA genes),comparisons of the diversity of bacteria associated withPST producing dino£agellate strains, A. lusitanicumNEPCC 253 and A. tamarense NEPCC 407 [11^13] andthose of A. tamarense PCC 173a and Scrippsiella trochoi-dea NEPCC 15 which have no previously reportedlink with toxicity [14], were made. Furthermore compar-isons were made with bacterial isolates obtained fromAlexandrium spp. cultures 4 years prior to this investiga-tion [6].

2. Materials and methods

2.1. Dino£agellate cultures

The dino£agellates A. lusitanicum NEPCC 253,A. tamarense NEPCC 407, A. tamarense PCC 173a andS. trochoidea NEPCC 15 were cultured in Guillards f/2without silicate (Sigma, Dorset, UK) enriched seawater[15]. Cultures were maintained at 15³C with a 14:10 h,light:dark cycle (irradiance level 0.5^1.5U1016 quantas31 cm32). Culture development was monitored daily bytriplicate cell counts using a Sedgwick^Rafter countingchamber and visualised by the addition of Lugol's iodine[16]. NEPCC strains were supplied by the North EastPaci¢c Culture Collection, British Columbia, Canadaand A. tamarense PCC 173a was from the Plymouth Cul-ture Collection, Plymouth, UK.

2.2. Isolation of bacteria from dino£agellate cultures

Samples (1 ml) of dino£agellate cultures from early, lateexponential and stationary growth phases were seriallydiluted (10-fold dilutions; neat ^ 1039) in sterile seawater.Aliquots (100 Wl) of each dilution were spread, in tripli-cate, onto marine agar medium (Difco 2216, Michigan,USA) and subsequently incubated for 14 days at 20³C.Bacteria from the dilution containing between 50 and100 colonies were isolated from one of the replicate platesand replated individually onto marine agar, to obtain purecultures. Resultant bacterial isolates were categorised bycolony morphology and examined microscopically fortheir reaction to Gram stain. Isolates were stored in ma-rine broth plus 10% (v/v) glycerol at 370³C, prior toRFLP investigation.

2.3. RFLP analysis

Bacteria isolated from dino£agellate cultures were resus-pended in ATL bu¡er (Qiagen, Dorking, UK) and ge-nomic DNA from each isolate was extracted followingthe QIAmp tissue protocol (Qiagen, Dorking, UK).DNA was stored at 320³C prior to PCR ampli¢cation.Small subunit (SSU) 16S rDNA was PCR-ampli¢ed fromtotal genomic DNA, using the universal eubacterial prim-ers 27F and 1522R (Bioline, London, UK) [17]. In a ¢nalvolume of 50 Wl, the reaction mixture contained 100 pmolof each ampli¢cation primer, 100 WM each deoxynucleo-side triphosphate, 1.5 mM MgCl2, 10 ng template and 5 Uof Taq DNA polymerase (Bioline, London, UK), whichwas added after a pre-cycling step in which the reactionmixture was heated to 95³C for 5 min. Ampli¢cation con-ditions were 95³C for 1 min, 55³C for 1 min, and 72³C for1 min, for a total of 35 cycles followed by 72³C for 10 min[17]. Ampli¢cation products were visualised by electropho-resis using a 2% (w/v) agarose gel (Sigma, Dorset, UK)

FEMSEC 1273 9-10-01

G.L. Hold et al. / FEMS Microbiology Ecology 37 (2001) 161^173162

containing ethidium bromide (0.5 mg ml31), with a 1-kbmarker (Gibco, Glasgow, UK) included on the gel forreference.

To generate RFLP patterns, approx. 700 ng of eachPCR product was digested with 5 units of the restrictionendonuclease HaeIII (Promega, Southampton, UK), fol-lowing the manufacturer's speci¢cations. Reactions werestopped by the addition of EDTA, and fragments wereresolved by gel electrophoresis in 2.5% low melting pointagarose gel (Gibco, Glasgow, UK), containing ethidiumbromide (0.5 g ml31). DNA fragments were visualisedunder UV illumination with RFLP patterns grouped usingPhoretix ID v 4 computer software (Phoretix Internation-al, Newcastle upon Tyne, UK).

2.4. DGGE analysis of 16S rDNA fromdino£agellate cultures

Cell pellets from 1-l cultures, at early, late exponentialand stationary growth phases, of A. lusitanicum NEPCC253, A. tamarense NEPCC 407 and PCC 173a andS. trochoidea NEPCC 15 were obtained by centrifugationat 10 000Ug for 10 min. The cell pellet was resuspended inTE bu¡er (1 ml) and stored at 320³C until extraction ofDNA. Following thawing, glass beads (0.5 g) of 0.16^1.17mM diameter (Sigma, Dorset, UK) were added to thesamples, which were alternately vortexed and placed onice for 60 s, until visual signs of dino£agellate cell lysiswere observed. Following addition of 50 Wl lysozyme(50 mg ml31 in distilled water) (Sigma, Dorset, UK),samples were incubated for 30 min at room temperature.Sodium dodecyl sulfate (SDS) (100 Wl, 10% (w/v) in dis-tilled water) (Sigma, Dorset, UK) was mixed with eachsample, followed by addition of 50 Wl proteinase K(20 mg ml31 in distilled water) (Sigma, Dorset, UK) andfurther incubation at room temperature for 30^60 minbefore phenol/chloroform extraction [18].

Bacterial 16S rDNA was ampli¢ed using primers de-scribed by Muyzer et al. [19] which generated ampli¢-cation products corresponding to nucleotide positions341^534 in Escherichia coli. Ampli¢cation conditionswere 94³C for 30 s, 55³C for 45 s, and 72³C for 30 s,for 30 cycles followed by 72³C for 10 min. Ampli¢ca-tion products were visualised by electrophoresis usinga 2% (w/v) agarose gel (Sigma, Dorset, UK) con-taining ethidium bromide (0.5 mg ml31), with a 100-bpmarker (Gibco, Glasgow, UK) included on the gel forreference.

DGGE was performed with a D-code 16/16 cm gel sys-tem using a 1.0 mm gel width (Bio-Rad, Herts, UK) main-tained at a constant temperature of 65³C in 6 l of 1UTAEbu¡er (20 mM Tris acetate, 0.5 mM EDTA, pH 8.0).Gradients were formed between 20 and 60% denaturant(with 100% denaturant de¢ned as 7 M urea and 40% (v/v)formamide). Gels were run at 130 V for 4^6 h and stained

in 1UTAE containing ethidium bromide (0.5 mg l31) be-fore destaining in distilled water for 15 min. Pro¢les wereinspected under UV illumination.

DGGE bands were excised using a sterile razor bladeand DNA eluted overnight in 100 Wl of TE bu¡er. DNAwas recovered using the Wizard PCR prep DNA puri¢ca-tion system (Promega, Southampton, UK), with 5-Wl ali-quots of DNA from each excised band used as template inPCR ampli¢cations as described above. PCR productswere puri¢ed using Qiagen PCR spin columns (Qiagen,Dorking, UK).

2.5. 16S rDNA sequence determination andphylogenetic analysis

Representative bacterial isolates, from dino£agellatecultures, possessing unique colony morphologies orRFLP patterns and bacteria previously isolated from di-no£agellate strains A. tamarense UW2c, A. tamarenseUW4, A. tamarense NEPCC 407, A. lusitanicum NEPCC253 and Alexandrium a¤ne NEPCC 667 [6] were selectedfor DNA sequence analysis. Several bacterial isolates fromeach group were partially sequenced in order to con¢rmcolony morphology and RFLP analyses were e¡ective atclassifying the cultured isolates. However, only one repre-sentative sequence from each group, spanning nearly theentire 16S rRNA gene, was submitted to the database. 16SrRNA gene PCR amplicons were puri¢ed using Qiaquick-spin columns (Qiagen, Dorking, UK) and subsequentlysequenced bidirectionally, using universal 16S rRNAprimers [20]. PCR amplicons obtained from DGGE pro-¢les were also sequenced bidirectionally, using primers de-scribed by Muyzer et al. [19], on a 373A automated DNAsequencer using Taq polymerase initiated cycle sequencingwith £uorescent dye-labelled dideoxynucleotides with pro-tocols recommended by the manufacturer (Perkin Elmer,Bucks, UK). Nucleotide sequence fragments generatedwere assembled into consensus sequences using the GCGpackage v 7.0-UNIX [21].

The automatic alignment function of the ARB sequenceanalysis software package [22] was used initially to alignthe 16S rRNA gene sequences with reference sequencesobtained from GenBank [23], the Ribosomal DatabaseProject (RDP) [24], and the ARB database (Departmentof Microbiology, Technical University of Munich, Mu-nich, Germany). The Genetic Data Environment (GDE)v 2.2 [25] sequence analysis software package was usedsubsequently to re¢ne the alignment generated by ARBmanually. Pairwise sequence similarities were generated,using GDE, and individual sequence masks employedfor each pair of sequences analysed.

2.6. Nucleotide sequence accession numbers

Nucleotide sequences from the cultured bacteria were

FEMSEC 1273 9-10-01

G.L. Hold et al. / FEMS Microbiology Ecology 37 (2001) 161^173 163

¢led in GenBank under the following accession numbers:253-13 (AJ294351), 253-16 (AJ294352), 667-2 (AJ294353),667-4 (AJ294354), 667-19 (AJ294355), 667-12 (AJ294356),4KVS17 (AJ294358), 407-13 (AJ294359), 407-2(AJ294360), 2C3 (AJ294361), 253-19 (AJ294362), 667-16(AJ296288), ALUS253_28 (AF359524), ATAM407_61(AF359525), ALUS253_43 (AF359526), ALUS253_25(AF359527), ATAM407_68 (AF359528), ATAM407_18(AF359529), ATAM407_62 (AF35930), ATAM173a_16(AF359531), ATAM173a_17 (AF359532), ATAM407_58(AF359533), SCRIPPS_732 (AF359534), ATAM407_56(AF359535), SCRIPPS_101 (AF359537), ATAM173a_51(AF359538), ATAM173a_2 (AF359539), ATAM173a_3(AF359540), ATAM173a_6 (AF359541), ATAM173a_5(AF359542), ATAM173a_36 (AF359543), ATAM173a_9(AF359544), SCRIPPS_94 (AF359545), SCRIPPS_739(AF359546), SCRIPPS_423 (AF359547), SCRIPPS_413(AF359548), SCRIPPS_426 (AF359549), SCRIPPS_740(AF359550), ALUS253_6 (AF359551), ATAM407_2035(AF359552).

3. Results

3.1. Diversity of culturable bacteria

Table 1 lists the 16S rDNA sequences most closely re-lated to those of the 12 bacterial strains previously isolatedfrom stationary phase batch cultures of Alexandrium spp.by Gallacher et al. [6]. All strains examined were clearlyplaced in the K- and Q-Proteobacteria subphyla, with theexception of strain 667-16, which was Gram-positive andmost closely related to Micrococcus luteus. Other bacteriaisolated from the weakly toxic A. a¤ne NEPCC 667 be-longed to the Roseobacter clade. Some bacteria isolatedfrom A. lusitanicum NEPCC 253 were also associatedwith the Roseobacter clade and showed high sequence sim-ilarities with bacterial 16S rRNA gene clones recoveredfrom a culture of the toxic dino£agellate P. lima [4]. How-ever, these strains were found to be related only distantlyto the original Roseobacter algicola (now Ruegeria algico-la) isolated from the phycosphere of P. lima (data not

Fig. 1. Distribution of bacteria associated with the three growth phases of the dino£agellates A. lusitanicum NEPCC 253, A. tamarense NEPCC 407, A.tamarense PCC 173a and S. trochoidea NEPCC15. Results are expressed as mean values from triplicate analyses þ S.D.

FEMSEC 1273 9-10-01


shown). The 16S rDNA sequences of other bacterialstrains isolated from A. lusitanicum, A. tamarense NEPCC407 and UW2c, possessed high sequence similarities withthose of the Q-Proteobacterium Alteromonas macleodii, acommon inhabitant of coastal water and open oceans [26],and Marinobacter aquaeolei, originally isolated from theNorth Sea [27].

All bacteria isolated from Alexandrium spp. and S. tro-choidea cultures in this investigation were identi¢ed asGram-negative rods, with the majority of isolates (90%)

forming distinct non-pigmented colonies ; other bacteriaexhibited pink, brown and yellow pigmentation followinggrowth on marine agar (Table 2). Using colony morphol-ogy and RFLP pro¢les, the cultured bacteria were cate-gorised into 26 groups (Fig. 1, Table 2). Based on 16SrDNA sequence analysis of representative isolates fromeach RFLP group, bacteria were identi¢ed as belongingto the K-Proteobacteria (with many sequences falling with-in the Roseobacter clade), Q-Proteobacteria and the Cyto-phaga-Flavobacter-Bacteroides (CFB) phylum (Table 2).

Table 116S rDNA sequence comparison of strains previously isolated from toxic Alexandrium spp. cultures, with 16S rDNA database sequence entries

Bacterialstrain

Phylogenetic group Most closely related sequencefrom a validly describedbacterial species (GenBankaccession No.)a

Sequencesimilarity(%)

Most closely related databasesequence (GenBank accessionNo.)b

Sequencesimilarity(%)

A. a¤ne NEPCC 667667-2 K-Proteobacteria (Roseobacter

clade)Roseobacter gallaeciensis (Y13244) 96.9 ATAM407_56 100

667-4 K-Proteobacteria (Roseobacterclade)

Staleya guttiformis (Y16427) 99.2 ATAM173a_16 100


Antarctobacter heliothermus(Y115522)

99.4 ATAM173a_51 100


Sul¢tobacter pontiacus (Y13155) 95.7 env.PRLIST06 (Y15343) geneclone recovered fromProrocentrum lima

100

667-16 Gram-positive Micrococcus luteus (AJ296288) 98.6 ^* ^A. tamarense NEPCC 407407-2 Q-Proteobacteria Alteromonas macleodii (L10938) 98.7 ATAM407_18 100407-13 Q-Proteobacteria Marinobacter aquaeolei

(AF173969)99.2 ATAM407_2035 99.8

A. lusitanicum NEPCC 253253-13 K-Proteobacteria (Roseobacter

clade)Rosevarius tolerans (Y11551) 96.4 ATAM407_61 100

env. kpc 34 (AF194397) geneclone recovered from Aureococcusanophage¡erens

99.9

env.PRLISY03 (Y15348) geneclone recovered fromProrocentrum lima

99.7


Ruegeria gelatinovora (D88523) 96.8 env.PRLISY01 (Y15346) geneclone recovered fromProrocentrum lima

99.9

env.PRLIST02 (Y15339) geneclone recovered fromProrocentrum lima

99.9

env.kpc101f (AF194394) geneclone recovered from Aureococcusanophage¡erens

99.9

253-19 Q-Proteobacteria Alteromonas macleodii (L10938) 98.3 ^ ^A. tamarense UW2c2c3 Q-Proteobacteria Alteromonas macleodii (L10938) 98.3 Alteromonas macleodii related

isolate CH-516 (Y18234)99.6

endocytic bacterium Noc15(AF262741)

99.4

A. tamarense UW4c4Kvs17 K-Proteobacteria Aquaspirillum itersonii (Z29620) 89.5 endocytic bacterium Noc17

(AF262750)99.5

^* indicates validly published sequence showed highest sequence similarity.aMost closely related validly published sequence indicates the de¢ned type strain with highest 16S rDNA sequence similarity to each particular isolate.bMost closely related database sequence indicates the 16S rDNA sequence within the database which has highest 16S rDNA sequence similarity to eachparticular isolate.

FEMSEC 1273 9-10-01


Tab

le2

Cha

ract

eris

atio

nof

bact

eria

isol

ated

onm

arin

eag

ar,

from

the

four

dino

£age

llate

cult

ures

A.

lusi

tani

cum

NE

PC

C25

3,A

.ta

mar

ense

NE

PC

C40

7,A

.ta

mar

ense

PC

C17

3aan

dS

.tr

ocho

idea

NE

PC

C15

RF

LP

patt

ern

Col

ony

mor

phol

ogy

Rep

rese

ntat

ive

isol

atea

Num

ber

ofsi

mila

rba

cter

iase

quen

ced

Mos

tcl

osel

yre

late

dse

quen

cefr

oma

valid

lyde

scri

bed

bact

eria

lsp

ecie

s(G

enB

ank

acce

ssio

nN

o.)b

Sequ

ence

sim

ilari

ty(%

)

Mos

tcl

osel

yre

late

dda

taba

sese

quen

ce(G

enB

ank

acce

ssio

nN

o.)c

Sequ

ence

sim

ilari

ty(%

)

K-P

rote

obac

teri

a1

circ

ular

,sm

ooth

surf

ace,

umbo

nate

,cr

eam

AL

US2

53_2

87

Ahr

ensi

aki

elen

se(D

8852

4)98

.5^*

^

2ir

regu

lar,

wit

hlo

bate

mar

gin,

crea

mA

LU

S253

_25

1S

tapp

iast

ellu

lata

(D88

525)

98.6

PT

B1

(Y10

913)

99.7

PT

B5

(AJ0

0064

6)99

.43

circ

ular

,sm

ooth

,ra

ised

,cr

eam

AT

AM

407_

681

Phy

lloba

cter

ium

myr

sina

cear

um(D

1278

9)96

.8en

v.R

SHD

3S10

(AF

1902

14)

gene

clon

efr

omP

¢est

eria

dino

£age

llate

cult

ure

CC

MP

1829

97.9

env.

HN

SS21

from

mar

ine

spon

ge(Z

8857

2)97

.0

4ir

regu

lar

wit

hun

dula

tem

argi

n,co

nvex

,br

own

cent

rew

ith

crea

mm

argi

n

SCR

IPP

S_94

3A

quas

piri

llum

iter

soni

i(Z

2962

0)89

.5en

docy

tic

bact

eriu

mN

oc17

(AF

2627

50)

99.5

4Kvs

17(s

eeT

able

1)99

.15

circ

ular

,en

tire

,co

nvex

,sm

ooth

,br

own

cent

rew

ith

crea

mm

argi

nSC

RIP

PS_

739

2H

ypho

mon

asjo

hnso

nii

(AF

0827

91)

98.7

^^

6ci

rcul

ar,

pulv

inat

e,en

tire

,sm

ooth

,w

hite

cent

rew

ith

crea

mm

argi

nSC

RIP

PS_

423

2H

ypho

mon

asoc

eani

tis

(AF

0827

97)

90.9

uncu

ltur

edba

cter

ium

BU

RT

ON

-39

(AF

1428

57)

96.3

7ci

rcul

ar,

pulv

inat

e,sm

ooth

,cr

eam

SCR

IPP

S_42

61

Cau

loba

cter

vibr

ioid

es(A

J227

754)

93.7

uncu

ltur

edC

aulo

bact

er5C

2(A

F24

5033

)94

.8

K-P

rote

obac

teri

a(R

oseo

bact

ercl

ade)

8ci

rcul

ar,

conv

ex,

crea

mw

ith

light

pink

cent

reA

LU

S253

_13

Rue

geri

aal

gico

la(X

7831

3)94

.525

3-13

(see

Tab

le1)

99.9

env.

PR

LIS

Y03

(Y15

348)

gene

clon

ere

cove

red

from

Pro

roce

ntru

mlim

a99

.7

AT

AM

407_

612

Ros

eova

rius

tole

rans

(Y11

551)

96.5

env.

kpc

34(A

F19

4397

)ge

necl

one

reco

vere

dfr

omA

ureo

cocc

usan

opha

ge¡

eren

s

99.9

9ci

rcul

ar,

conv

ex,

crea

mw

ith

light

pink

cent

reA

LU

S253

_43

10R

oseo

vari

usto

lera

ns(Y

1155

1)99

.6^

^

AT

AM

407_

254

asab

ove

10ir

regu

lar

wit

han

undu

late

mar

gin,

smoo

th,

conv

ex,

dark

brow

nA

TA

M40

7_62

2O

ctad

ecab

acte

ran

tarc

ticu

s(U

1458

3)94

.3en

v.D

049

(AF

1775

68)

gene

clon

ere

cove

red

from

seaw

ater

95.5

env.

EK

H01

8(A

F14

2900

)ge

necl

one

reco

vere

dfr

omse

awat

er95

.4

env.

MC

-1(A

F18

8164

)ge

necl

one

from

P¢e

ster

iasp

.95

.4

11ir

regu

lar

wit

hlo

bate

mar

gin,

conv

ex,

smoo

th,

brow

nce

ntre

AT

AM

407_

581

Sul

¢tob

acte

rm

edit

erra

neus

(Y17

387)

98.7

^10

0

AT

AM

173a

_16

1S

tale

yagu

ttif

orm

is(Y

1642

7)99

.266

7-4

(see

Tab

le1)

^A

TA

M17

3a_1

71

Sul

¢tob

acte

rm

edit

erra

neus

(Y17

387)

99.7

^99

.9A

TA

M17

3a_4

93

Sul

¢tob

acte

rpo

ntia

cus

(Y13

155)

96S

ul¢t

obac

ter

sp.

DSS

-2(A

F09

8490

)99

.9SC

RIP

PS_

732

2S

ul¢t

obac

ter

pont

iacu

s(Y

1315

5)99

.8S

ul¢t

obac

ter

sp.

DSS

-2(A

F09

8490

)10

0

FEMSEC 1273 9-10-01


Tab

le2

(con

tinu

ed)

RF

LP

patt

ern

Col

ony

mor

phol

ogy

Rep

rese

ntat

ive

isol

atea

Num

ber

ofsi

mila

rba

cter

iase

quen

ced

Mos

tcl

osel

yre

late

dse

quen

cefr

oma

valid

lyde

scri

bed

bact

eria

lsp

ecie

s(G

enB

ank

acce

ssio

nN

o.)b

Sequ

ence

sim

ilari

ty(%

)

Mos

tcl

osel

yre

late

dda

taba

sese

quen

ce(G

enB

ank

acce

ssio

nN

o.)c

Sequ

ence

sim

ilari

ty(%

)

12ci

rcul

arw

ith

enti

rem

argi

n,sm

ooth

,cr

eam

AT

AM

407_

567

Ros

eoba

cter

galla

ecie

nsis

(Y13

244)

96.9

667-

2(s

eeT

able

1)10

0

circ

ular

wit

her

ose

mar

gin,

gran

ular

,um

bona

tew

ith

abr

own

cent

rean

dpa

lem

argi

n

SCR

IPP

S_10

12

Sul

¢tob

acte

rbr

evis

(Y16

425)

97.2

Sul

¢tob

acte

rsp

.D

SS-2

(AF

0984

90)

97.3

13ci

rcul

ar,

smoo

th,

gela

tino

us,

rais

ed,

crea

mA

TA

M17

3a_5

12

Ant

arct

obac

ter

helio

ther

mus

(Y11

552)

99.6

667-

12(s

eeT

able

1)10

0

14pu

ncti

form

,ge

lati

nous

,pi

nkA

TA

M17

3a_9

2R

oseo

vari

usto

lera

ns(Y

1155

1)96

.3en

v.P

RL

ISY

03(Y

1534

8)ge

necl

one

reco

vere

dfr

omP

roro

cent

rum

lima

98.3

Q-P

rote

obac

teri

a15

circ

ular

,sm

ooth

,ge

lati

nous

,£a

t,cr

eam

AT

AM

407_

183

Alt

erom

onas

mac

leod

ii(X

8214

5)98

.940

7-2

(see

Tab

le1)

99.9

2c3

(see

Tab

le1)

97.9

253-

19(s

eeT

able

1)97

.716

circ

ular

,co

nvex

,ge

lati

nous

,ye

llow

AT

AM

173a

_51

Alt

erom

onas

mac

leod

ii(A

F17

3965

)93

.7po

lar

gas

vacu

olat

est

rain

214.

6(U

7372

4)97

.1

17ci

rcul

arm

ucoi

d,ge

lati

nous

,la

rge

crea

mA

TA

M40

7_20

352

Mar

inob

acte

raq

uaeo

lei

(AF

1739

69)

99.3

Mar

inob

acte

rsp

.D

S40M

8(A

F19

9440

)99

.8

407-

13(s

eeT

able

1)99

.8A

TA

M17

3a_3

61

Pse

udoa

lter

omon

asha

lopl

ankt

issu

bsp.

tetr

aodo

nis

(AF

2147

30)

99.8

^^

18ir

regu

lar,

loba

te,

£at,

yello

wSC

RIP

PS_

740

1P

seud

omon

asst

utze

ri(U

2641

5)99

.2un

cult

ured

gam

ma

prot

eoba

cter

ium

(AB

0108

54)

99.4

Cyt

opha

ga-F

lavo

bact

er-B

acte

roid

es(C

FB

)19

circ

ular

,ge

lati

nous

,ra

ised

,br

ight

yello

wA

TA

M17

3a_2

2P

olar

ibac

ter

irge

nsii

(M61

002)

96.6

EB

AC

34(A

F26

8227

)ge

necl

one

isol

ated

from

seaw

ater

98.0

AT

AM

173a

_33

Gel

idib

acte

ral

gens

(U62

914)

)92

.3m

arin

eps

ychr

ophi

leSW

17(A

F00

1368

)94

.6

20ci

rcul

ar,

£at,

yello

wA

TA

M17

3a_6

1C

ellu

loph

aga

ulig

inos

a(M

6279

9)93

.7C

ytop

haga

sp.

KT

02ds

18(A

F23

5126

)98

.1

21ci

rcul

ar,

conv

ex,

smoo

th,

crea

mSC

RIP

PS_

413

1P

sych

rose

rpen

sbu

rton

ensi

s(U

6291

3)93

.1F

lavo

bact

eriu

msp

.5N

-3(A

B03

2514

)94

.822

circ

ular

,en

tire

,ra

ised

,ge

lati

nous

,ye

llow

AL

US2

53_6

2G

elid

ibac

ter

alge

ns(A

F00

1367

)92

.0C

ytop

haga

sp.

(AB

0170

46)

96.5

^*in

dica

tes

valid

lyde

scri

bed

sequ

ence

show

edhi

ghes

tse

quen

cesi

mila

rity

.aR

epre

sent

ativ

eis

olat

esw

ere

obta

ined

from

:A

LU

S_25

3,A

.lu

sita

nicu

mN

EP

CC

253

;A

TA

M_4

07,

A.

tam

aren

seN

EP

CC

407

;A

TA

M_1

73a,

A.

tam

aren

seP

CC

173a

;SC

RIP

PS,

S.

troc

hoid

eaN

EP

CC

15.

bM

ost

clos

ely

rela

ted

sequ

ence

from

ava

lidly

desc

ribe

dba

cter

ial

spec

ies

indi

cate

sth

ede

¢ned

type

stra

inw

ith

high

est

16S

rDN

Ase

quen

cesi

mila

rity

toea

chpa

rtic

ular

isol

ate.

c Mos

tcl

osel

yre

late

dda

taba

sese

quen

cein

dica

tes

the

16S

rDN

Ase

quen

cew

ithi

nth

eda

taba

sew

hich

has

high

est

16S

rDN

Ase

quen

cesi

mila

rity

toea

chpa

rtic

ular

isol

ate.

FEMSEC 1273 9-10-01


In general, each colony morphotype was associated witha unique HaeIII RFLP pattern. Exceptions to this obser-vation were bacterial isolates from Alexandrium culturesdescribed as circular, convex and cream-coloured with arose centre, which gave two distinct RFLP patterns (pat-terns 8 and 9) (Table 2). In contrast, examination of 16SrDNA sequence data from representative isolates fromA. tamarense NEPCC 407 and S. trochoidea cultures, cat-egorised as possessing RFLP pattern 12 but showing dif-ferent colony morphologies, indicated diverse phylogeneticneighbours (Table 2). Representative sequences fromRFLP patterns 11, 17 and 19 sharing the same colonycharacteristics also had distant phylogenetic neighbours.

3.2. Bacterial diversity associated with A. lusitanicumNEPCC 253

In this study, A. lusitanicum NEPCC 253 was observedto support the most limited culturable bacterial popula-tion, with bacterial isolates, characterised into ¢ve RFLPpatterns (patterns 1, 2, 8, 9 and 22), being prominent at allstages of sampling (Fig. 1). Sequence analysis of 16SrDNA from representative isolates indicated that the ma-jority of the bacteria associated with this dino£agellatewere K-Proteobacteria. RFLP pattern 8 and 9 isolateswhich grouped within the Roseobacter clade were closelyrelated with only a 2.5% di¡erence in their 16S rDNAsequence over the 1250 base pairs analysed. Bacteria ex-hibiting RFLP pattern 22 with strongest sequence similar-ity to Gelidibacter algens were the only isolates from thisdino£agellate to be located outside the K-Proteobacteriasubphylum (Table 2).

3.3. Bacteria associated with A. tamarense NEPCC 407

As observed in A. lusitanicum, bacteria generatingRFLP patterns 8 and 9 were present at all stages ofgrowth in cultures of A. tamarense NEPCC 407 but atmuch lower numbers (Fig. 1). However, in this culture,bacteria possessing pattern 15, with strong sequence sim-ilarity to the previously isolated 407-2, 2c3 and 253-19(Table 1), and RFLP pattern 12 isolates were also consis-tently present. Pattern 11 isolates were detected only atlate exponential and stationary growth phases of this cul-ture but at low levels (Fig. 1). Other K-Proteobacterialisolates possessing 16S rDNA sequence types formingRFLP patterns 3 and 10 only reached detectable levelsin the stationary growth phase of A. tamarense NEPCC407. Q-Proteobacterial isolates possessing 16S rDNA se-quence types showing banding pattern 17 reached onlylow levels in late exponential samples (Fig. 1), with 16SrRNA gene sequences from these bacteria(ATAM407_2035) almost identical to that of isolate 407-13 (Table 1) and also showing high similarities to 16SrDNA sequences obtained from environmental clone li-braries of S. trochoidea [28].

3.4. Bacteria associated with A. tamarense PCC 173a

Bacteria exhibiting RFLP patterns 8, 11, 13, 14, 16, 19and 20 were consistently present at all growth points ofA. tamarense PCC 173a, while pattern 17 isolates weredetected only during the exponential phase (Fig. 1, Table2). RFLP patterns 13, 16, 19 and 20 were characteristic ofisolates from A. tamarense PCC 173a in the current studywith the representative sequence from pattern 13 identi¢edas an K-Proteobacterium with 100% sequence similarity tothe previously isolated strain 667-12 (Table 1). The pattern16 representative belonged to the Q-Proteobacteria subphy-la and patterns 19 and 20 clustered within the CFB phy-lum.

3.5. Bacteria associated with S. trochoidea NEPCC 15

Of the dino£agellates studied, the culturable bacteriaassociated with S. trochoidea showed the highest degreeof phylogenetic variation over the course of the dino£a-gellate growth cycle (Fig. 1), with 12 di¡erent RFLP pat-terns identi¢ed. Following analysis of the sequence datafrom RFLP pattern representatives, isolates identi¢ed aspattern 12 were clearly distinguished from those bacteriacategorised as pattern 12 from A. tamarense NEPCC 407.Some of the bacteria had 16S rDNA sequences similar oridentical to those obtained from environmental clones ofthis dino£agellate (patterns 4 and 21) [28] or isolate4Kvs17, again previously obtained from Alexandriumspp. (pattern 4) (Tables 1 and 2).

On continued subculture, isolates showing RFLP pat-terns 23^26 could not be maintained and subsequently 16SrDNA phylogenetic analysis was not possible.

3.6. DGGE analysis of dino£agellate cultures

Examination of PCR-ampli¢ed 16S rDNA DGGE pro-¢les demonstrated that di¡erent pro¢les were detected foreach dino£agellate culture (Fig. 2). Each pro¢le remainedconstant throughout the di¡erent growth phases althoughindividual band intensities varied during growth. In allcases, more DGGE bands were identi¢ed than culturedisolates, as distinguished by RFLP patterns and colonymorphologies (Figs. 1 and 2, Table 2). The PCR productsderived from total genomic DNA from A. lusitanicumNEPCC 253 and A. tamarense NEPCC 407 each generated10 distinct DGGE bands, while those of A. tamarensePCC 173a and S. trochoidea NEPCC 15 generated 16bands (Fig. 2). 16S rDNA DGGE bands were excisedfor sequence analysis although some bands failed to re-amplify or contained mixed sequences, so further analysiswas not possible. Thus, overall, DGGE pro¢ling did notdetect the same diversity of bacteria which could be cul-tured on marine agar. In A. lusitanicum NEPCC 253,A. tamarense NEPCC 407 and PCC 173a, identically posi-tioned bands from the di¡erent growth points were excised

FEMSEC 1273 9-10-01


to assess reproducibility in the DGGE pro¢les from di¡er-ent growth samples. The sequences recovered from theseco-migrating bands from the di¡erent sampling points(early and late exponential and stationary), were virtuallyidentical with di¡erences observed assumed to be artifactsdue to single sequencing reactions.

3.7. 16S rDNA DGGE analysis of A. lusitanicumNEPCC 253

A representative cultured isolate from each of the ¢veRFLP patterns obtained from A. lusitanicum NEPCC 253was successfully ampli¢ed using the PCR conditions forDGGE analysis and demonstrated 100% sequence recog-nition with the primers. However, DGGE analysis of thisdino£agellate culture failed to identify the presence of allRFLP groups. 16S rDNA sequence analysis of excisedDGGE bands in A. lusitanicum cultures demonstratedthe presence of members of the Roseobacter clade (Fig.2, bands 253/6, 7, 10) with strong sequence similarity toRFLP pattern 3 and other K-Proteobacteria most closelyrelated to SCRIPPS_739 ^ a Hyphomonas-related isolate(Fig. 2, 253/2, 4). A further band (253/3) was related to theCytophagales and possessed an identical 16S rDNA se-quence to that of ALUS253_6 (RFLP pattern 22) andG. algens (AF001367). Proteobacteria from the Q-subphy-lum were not cultured from this dino£agellate at anygrowth phase examined and, subsequently, 16S rDNA se-quences belonging to this group were also not recoveredfrom DGGE gels (Table 3).

3.8. 16S rDNA DGGE analysis of A. tamarenseNEPCC 407

As with A. lusitanicum NEPCC 253, all cultured isolatesshowed 100% sequence similarity to DGGE primers, with

representatives from each RFLP pattern identi¢ed fromthis dino£agellate amplifying under the PCR conditionsused for DGGE analysis, although again not all RFLPpatterns were represented in the community pro¢les. 16SrDNA DGGE analysis of A. tamarense NEPCC 407 in-dicated the presence of many Roseobacter-related sequen-ces with six bands excised from di¡erent positions in theDGGE pro¢le showing greater than 98% sequence simi-larity to Roseobacter-related bacterial 16S rDNA sequen-ces over the approx. 200 nucleotides analysed (Fig. 2).Roseobacter-related cultured isolates classi¢ed as belong-ing to RFLP patterns 9, 11 and 12 were all identi¢edwithin the DGGE pro¢le with s 99% sequence similarity.Other bacterial 16S rDNA sequences identi¢ed from theDGGE pro¢les included a Hyphomonas-related sequence(isolate SCRIPPS_739 (97.7%)), within the K-Proteobacte-ria (band 407/3). However, K-Proteobacteria, excludingRoseobacter strains, were not isolated by culturing at earlygrowth phases of this dino£agellate. Other DGGE 16SrDNA bands possessed low similarities (6 90%) to Q-Pro-teobacteria (407/4) and an unknown marine psychrophilewithin the CFB phylum (407/2), although CFB phylumbacteria were not detected from this dino£agellate cultureusing enrichment techniques.

3.9. 16S rDNA DGGE analysis of A. tamarense PCC 173a

The majority of 16S rDNA DGGE bands excised werecon¢rmed as Roseobacter-related sequences. This resultcorresponds with the observation of the high percentageof these bacteria cultured from this dino£agellate on ma-rine agar medium (Figs. 1 and 2). However, other K-pro-teobacterial sequences detected by DGGE were not ob-served in this culture by cultivation on marine agar, forexample Sphingomonas sp., corresponding to band 173a/2(Fig. 2, Table 3). Also, con¢rmation of the presence of

Table 3Comparison of the percentage of cultured isolates classi¢ed as being Roseobacter related, K-Proteobacteria (not including Roseobacter-related isolates),Q-Proteobacteria and CFB and non-cultured (DGGE) pro¢les

Percent of isolates

A. lusitanicum NEPCC 253 A. tamarense PCC 407 A. tamarense PCC 173a S. trochoidea NEPCC 15

Earlyexp.

Lateexp.

Station-ary

Earlyexp.

Lateexp.

Station-ary

Earlyexp.

Lateexp.

Station-ary

Earlyexp.

Lateexp.

Station-ary

K-Proteo-bacteria(Roseobacter)

Cultured 66.7 52.3 30.8 55.7 76 70.9 43.5 66.7 64.2 33.3 31.3 25

DGGE 6 6 6 6 6 6 6 6 6 6 6 6

K-Proteo-bacteria

Cultured 16.7 4.1 30.8 ND ND 11.2 ND ND ND 66.7 62.4 50

DGGE 6 6 6 6 6 6 6 6 6 6 6 6

Q-Proteo-bacteria

Cultured ND ND ND 44.3 24 17.9 21.8 6.6 10.1 ND ND 25

DGGE ND ND ND 6 6 6 6 6 6 6 6 6

CFB Cultured 16.6 47.3 38.4 ND ND ND 34.7 26.7 25.7 ND 6.3 NDDGGE 6 6 6 6 6 6 6 6 6 6 6 6

6 denotes the presence of bacterial 16S rDNA detected sequences by DGGE analysis ; ND, not detected.

FEMSEC 1273 9-10-01


bacteria belonging to the CFB phylum was obtained fromDGGE (Fig. 2, bands 173a/12 and 16). Q-Proteobacteria-related sequences were also detected (band 173a/13), withother bands possessing low sequence similarity values todatabase 16S rDNA sequences (Fig. 2, ATAM173a/7).Examination of the 16S rDNA sequences obtained fromcultured isolates from A. tamarense PCC 173a indicatedthat bacterial isolates grouped as RFLP patterns 14, 16, 17and 19 did not have 100% sequence similarity with theDGGE primers and would therefore not be representedin the DGGE pro¢les. However, all other RFLP patternsfrom this algal culture were con¢rmed by DGGE analysis

3.10. 16S rDNA DGGE analysis of S. trochoideaNEPCC 15

Sequence determination and analysis of DGGE bandsexcised from pro¢les from the PCR-ampli¢ed 16S rDNAof S. trochoidea cultures was less successful, with onlylimited sequence information being generated. 16SrDNA DGGE pro¢les from Scrippsiella cultures, however,along with A. tamarense PCC 173a contained the highestnumber of bands (Fig. 2). As with A. lusitanicum andA. tamarense NEPCC 407, all cultured isolates showed100% sequence similarity with the DGGE primers,although only limited RFLP patterns were con¢rmed inthe DGGE analysis. The DGGE bands which could besequenced were a¤liated to all phyla in this alga detectedby culturing (Table 3). However, culturing on marine agarfailed to detect members of the Q-Proteobacteria in theexponential growth phase of the dino£agellate culture,although two adjacent bands from within the DGGE pro-¢le possessed between 94 and 96% sequence similarity toQ-Proteobacteria belonging to the Pseudomonas genus (Fig.1, RFLP pattern 25; Fig. 2, SCRIPP/6 and 7).

In contrast, DGGE band SCRIPP/13 possessed highsimilarity with the 16S rDNA sequences of isolates form-ing RFLP pattern 12 (ATAM407_56 and 667-2), culturedfrom this dino£agellate only in earlier growth phases.

4. Discussion

In this study, bacterial strains isolated 4 years previ-ously from a range of dino£agellate cultures were charac-terised by analysis of their 16S rRNA gene sequences (Ta-ble 1). Previous HPLC and CE-MS evidence suggestedthat bacterial isolate 667-2 belonging to the Roseobacterclade, and isolates 407-2 and 253-19 both closely related to

Fig. 2. DGGE pro¢les of stationary phase samples from the 16S rRNAgene V3 region of dino£agellate cultures A. lusitanicum NEPCC 253, A.tamarense NEPCC 407, A.tamarense PCC 173a and S. trochoideaNEPCC 15. Closest relative sequences are indicated for each band, withpercentage sequence identity noted. Bands classi¢ed as `poor sequence'indicate no closest relative sequence could be de¢ned.6

FEMSEC 1273 9-10-01


Am. macleodii, may be capable of synthesising multiplePSP derivatives [6]. Other putatively toxigenic bacteria in-clude isolate 253-13, also belonging to the Roseobacterclade, and isolate 2c3, again showing high similarity withAm. macleodii (Gallacher et al., unpublished). Thesestrains were associated with stationary phase cultures ofthe dino£agellate and it was considered to be of interest todetermine the predominance of bacterial strains, particu-larly those which are considered toxic, at di¡erent stagesof the dino£agellate life cycle. Hence we have further ex-tensively characterised and compared the bacterial consor-tia living with both toxic and non-toxic Alexandrium spe-cies and a dino£agellate species not associated withtoxicity (S. trochoidea). Toxin pro¢les of the dino£agel-lates strains A. lusitanicum NEPCC 253 and A. tamarenseNEPCC 407 used in this study have previously been in-vestigated on several occasions [10^13] and prior to thisstudy their toxicity was con¢rmed by HPLC (data notshown).

A combination of colony morphology and RFLP anal-ysis of PCR-ampli¢ed 16S rRNA genes was used to sortthe large number of cultivated isolates obtained from thedino£agellates into groups, thereby reducing the extent of16S rDNA sequence analysis. Previous work by Lebaronet al. [29] on marine bacteria demonstrated that colonymorphology underestimated taxonomic diversity. It wassuggested that, unless used in conjunction with other se-lection criteria, such as RFLP analysis of 16S rRNAgenes, e¡ective di¡erentiation between isolates could notbe achieved. In general, the majority of bacterial strainsisolated in the current study were distinguishable fromeach other using detailed colony morphology and RFLPindependently (Table 2) as bacterial strains sharing thesame RFLP pattern gave identical sequences (Table 2).Combining both techniques provided a powerful toolwhich proved useful in identifying strains for further phy-logenetic analysis.

Overall, the phylogenetic diversity was limited to twobacterial phyla, the Proteobacteria and CFB, with the for-mer restricted to the K- and Q-Proteobacteria subclasses.This is similar to the ¢ndings of Babinchak et al. [5] who,using oligonucleotide probes, found similar classes of bac-teria in a range of dino£agellates. Interestingly, compara-tive 16S rRNA gene sequence analyses revealed that manyof our isolates showed close identity to clades of predom-inantly marine bacterial isolates that do not contain val-idly described representative taxa. This result is commonin phylogenetic studies of naturally occurring marine bac-terial communities [28], and indicates that novel microbialspecies can be relatively easily cultivated from marine di-no£agellate cultures using standard marine agar media.

In this study, the K-Proteobacteria, particularly those ofthe Roseobacter clade, dominated the micro£ora of alldino£agellate cultures. One of these strains(ALUS253_25), isolated from A. lusitanicum, was foundto be closely related to PTB1 and PTB5 strains previously

isolated from a highly toxic Japanese A. tamarense clone[8]. Further, Roseobacter-related isolates from toxic A. lu-sitanicum and A. tamarense NEPCC 407 classi¢ed asRFLP pattern 8 demonstrated high sequence similaritywith the toxigenic bacterium 253-13, while pattern 12showed complete sequence identity with the sodium chan-nel blocking isolate 667-2 obtained by Gallacher et al. [6].

Many of the bacteria were related to microorganismswith known surface-associated life histories (CFB phylum,Hyphomonas, Caulobacter [30] and some members of theRoseobacter clade, for example Roseobacter algicola (re-classi¢ed as Ruegeria algicola [31] and found in the phyco-sphere of P. lima [32]), Roseobacter litoralis and Roseo-bacter denitri¢cans, obtained from the surface of greenseaweed [33]). This may be important in bacterial/dino£a-gellate interactions in relation to bacterial e¡ects on PSTproduction. One of the few studies to take into accountpossible e¡ects of bacterial adhesion on dino£agellatesdemonstrated a decrease in culture toxicity when toxigenicbacteria were prevented from physical contact with anaxenic A. lusitanicum culture [34].

Microbial cultivation also demonstrated that a dynamicsystem exists within dino£agellate culture, with shifts innumbers and/or composition of the bacterial £ora occur-ring throughout the dino£agellate vegetative cell cycle.However, PCR-DGGE ¢ngerprinting analyses of the mi-crobial community showed consistent pro¢les from vari-ous stages of the growth cycle. One possible explanationfor this discrepancy is that the active portion of the micro-bial community shifts throughout the dino£agellategrowth cycle, which is re£ected in the types of bacteriawe were able to culture at the di¡erent growth stages.Regardless of the reason, it is apparent that our studysu¡ers from the same cultivation biases that have beendocumented for other studies employing both microbialcultivation and cultivation-independent molecular ecologyapproaches. The DGGE analysis identi¢ed some gene se-quences which were not detected using the culture-basedmethod, for example the identi¢cation of K-Proteobacteriaoutwith the Roseobacter clade from A. tamarense PCC173a, thus highlighting the need to use both approaches(Table 3). In this study, the K-Proteobacteria, particularlythose of the Roseobacter clade, dominated the culturablemicro£ora of all dino£agellate cultures. A predominanceof Roseobacter-related sequences (with the exception ofS. trochoidea) was also identi¢ed by DGGE analysis,with more than 50% of sequences from the three Alexan-drium cultures being grouped within the clade.

Previously it was suggested that maximum bacterialnumbers occurred during stationary phase of the dino£a-gellate life cycle in batch culture, where dino£agellate celldeath could lead to increased availability of nutrients [3].This study indicated that the bacterial numbers £uctuatedaccording to cell type (as de¢ned by RFLP) independentlyof the dino£agellate growth phase.

Bacterial isolates from the non-toxin producing dino£a-

FEMSEC 1273 9-10-01


gellates S. trochoidea and A. tamarense PCC 173a showedthe most diverse populations with many of the strainsunique to these particular cultures. This suggests that aspecies-speci¢c association may exist between some bacte-ria and certain algal species and that di¡erences exist inthe micro£ora between toxic and non-toxic dino£agellates.However, further work using more dino£agellate culturesis required to determine if this is the case. Future studiesshould take into account the e¡ect on the isolated micro-£ora of using media other than marine agar and also thepotential of bacteria to resist cultivation because of theirinterdependence with other microbes [35].

Further work is also needed to ascertain if these bacte-rial populations are stable amongst di¡erent subcultures ofthe same dino£agellate strain. Spurious contamination ofalgal cultures in the laboratory cannot be discounted, butevidence demonstrating a functional signi¢cance of bacte-rial^algal associations (e.g. modulation of toxicity) wouldargue for a selective component to these relationships.This may indeed be the case as some bacterial strains,although isolated 4 years apart, were 99.9% similar basedon 16S rDNA sequence identity. This includes isolatesfrom the Alteromonas clade which were identical to thosepreviously reported as producing PST [6]. Also many ofthe bacterial gene sequences recovered from S. trochoideausing marine agar showed high phylogenetic similarity toenvironmental clone sequences obtained previously fromthe same culture [28].

In conclusion, this study has identi¢ed bacteria associ-ated with three growth phases of PST-associated dino£a-gellates, some of which are unde¢ned species/genera. Thestudy signi¢cantly adds to the limited information which iscurrently available within the sequence databases on bac-teria of algal origin. The results can also be used to inves-tigate the interaction of bacteria with dino£agellates inculture and in the environment. Speci¢cally, oligonucleo-tide probes for in situ hybridisation studies can now bedesigned to investigate the kinetics and physical associa-tion of these bacteria with dino£agellates in relation toboth toxicity and bloom dynamics.

Acknowledgements

We thank Jennifer Graham, Sharleen Alexander, PaulBurgess, Emma Collins and Faye Grant for their technicalassistance. This work was supported by Fisheries ResearchServices, Marine Laboratory, Aberdeen, and by EU grantFAIR CT96-1558. The work was also in part funded bythe European Community Human Capital and MobilityNetwork grant CHRX CT93-0194, and by the German^New Zealand Science and Technology Cooperation Proj-ect (NEU-014). The authors acknowledge the support ofProf. K.N. Timmis (Head of Division of Microbiology,National Research Centre for Biotechnology, Braun-schweig, Germany).

References

[1] Doucette, G.J. (1995) Assessment of the interaction of prokaryoticcells with harmful algal species. In: Toxic Marine Phytoplankton(Lassus, P., Arzul, G., Erard-Le Denn, E., Gentien P. and Marcail-lou-Le Baut, C., Eds.), pp. 385^394. Lavoisier, Paris.

[2] Doucette, G.J. (1995) Interactions between harmful algae and bacte-ria: a review. Nat. Toxins 3, 65^74.

[3] Gallacher, S. and Smith, E.A. (1999) Bacteria and paralytic shell¢shtoxins. Protist 150, 245^255.

[4] Prokic, I., Brummer, F., Brigge, T., Gortz, H.D., Gerdts, G., Schutt,C., Elbrachter, M. and Muller, W.E.G. (1998) Bacteria of the genusRoseobacter associated with the toxic dino£agellate Prorocentrumlima. Protist 149, 347^357.

[5] Babinchak, J.A., McGovern, E.R. and Doucette, G.J. (1998) Isola-tion and characterization of the bacterial £ora associated with PSP-related dino£agellate species. In: Harmful Algae (Reguera, B., Blan-co, J., Fernandez, M.L. and Wyatt, T., Eds.), pp. 410^413. Xunta deGalicia and Intergovernmental Oceanographic Commission ofUNESCO, Gra¢sant, Spain.

[6] Gallacher, S., Flynn, K.J., Franco, J.M., Brueggemann, E.E. andHines, H.B. (1997) Evidence for the production of paralytic shell¢shtoxins by bacteria associated with Alexandrium spp. (Dinophyta) inculture. Appl. Environ. Microbiol. 63, 239^245.

[7] Kodama, M., Ogata, T. and Sato, S. (1988) Bacterial production ofsaxitoxin. Agric. Biol. Chem. 52, 1075^1077.

[8] Kopp, M., Doucette, G.J., Kodama, M., Gerdts, G., Schut, C. andMedlin, L.K. (1997) Phylogenetic analysis of selected toxic and non-toxic bacterial strains isolated from the toxic dino£agellate Alexan-drium tamarense. FEMS Microbiol. Ecol. 24, 251^257.

[9] Groben, R., Doucette, G.J., Kopp, M., Kodama, M., Amann, R. andMedlin, L.K. (2000) 16S rRNA targeted probes for the identi¢cationof bacterial strains isolated from cultures of the toxic dino£agellateAlexandrium tamarense. Microb. Ecol. 39, 186^196.

[10] Hold, G.L., Smith, E.A., Birkbeck, T.H. and Gallacher, S. (2001)Comparison of paralytic shell¢sh toxin production by the dino£agel-lates Alexandrium lusitanicum NEPCC 253 and Alexandrium tama-rense NEPCC 407 in the presence and absence of bacteria. FEMSMicrobiol. Ecol. 36, 223^233.

[11] Cembella, A.D., Therriault, J.C. and Berland, P. (1988) Toxicity ofcultured isolates and natural populations of Protogonyaulax tama-rensis. Lebour, Taylor from the St. Lawrence Estuary. J. Shell¢shRes. 7, 611^622.

[12] Hummert, C., Donner, G., Reinhardt, K., Ritscher, M., Elbracher,M. and Luckas, B. (1998) Paralytic shell¢sh poisoning in Alexan-drium. A comparison of PSP patterns produced by twelve cultivatedstrains. In: Harmful Algae (Reguera, B., Blanco, J., Fernandez, M.L.and Wyatt, T., Eds.), pp. 397^401. Xunta de Galicia and Intergov-ernmental Oceanographic Commission of UNESCO, Gra¢sant,Spain.

[13] Mascarenhas, V., Alvito, P., Franca, S., Sousa, I., Martinez, A.G.and Rodriguez-Vazquez, J.A. (1995) The dino£agellate Alexandriumlusitanicum isolated from the coast of Portugal. Observations on tox-icity and ultrastructure during growth phases. In: Toxic Marine Phy-toplankton (Lassus, P., Arzul, G., Erard-Le Denn, E., Gentien, P.and Marcaillou-Le Baut, C., Eds.), pp. 71^76. Lavoisier, Paris.

[14] Schmidt, R.J. and Loeblich, A.R. (1979) A discussion of the system-atics of toxic Gonyaulax species containing paralytic shell¢sh poison-ing. In: Toxic Dino£agellate Blooms (Taylor, D.L. and Seliger, H.H.,Eds.), pp. 83^88. Elsevier/North-Holland, New York.

[15] Guillard, R.R.L. (1975) Culture of phytoplankton for feeding marineinvertebrates. In: Culture of Marine Invertebrate Animals (Smith,W.L. and Chanley, M.H., Eds.), pp. 29^60. Plenum Press, New York.

[16] McAlice, B.J. (1971) Phytoplankton sampling with the Sedgwick-Raf-ter cell. Limnol. Oceanogr. 16, 19^28.

[17] Suzuki, M., Rappe, M.S., Haimberger, Z.W., Win¢eld, H., Adair, N.,

FEMSEC 1273 9-10-01


Stro«bel, J. and Giovannoni, S.J. (1997) Bacterial diversity amongsmall-subunit rRNA gene clones and cellular isolates from thesame seawater sample. Appl. Environ. Microbiol. 63, 983^989.

[18] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Clon-ing, a Laboratory Manual, 2nd edn. Cold Spring Harbor LaboratoryPress, Plainview, NY.

[19] Muyzer, G., De Waal, E.C. and Uitterlinden, A.G. (1993) Pro¢ling ofcomplex microbial populations by denaturing gradient gel electro-phoresis analysis of polymerase chain reaction-ampli¢ed genes codingfor 16S rDNA. Appl. Environ. Microbiol. 59, 695^700.

[20] Lane, D.J. (1991) 16S/23S rRNA sequencing. In: Nucleic Acid Tech-niques in Bacterial Systematic (Stackebrandt, E. and Goodfellow, M.,Eds.), pp. 115^175. Wiley, New York.

[21] Devereux, J., Haeberli, P. and Smithies, O. (1984) A comprehensiveset of sequence analysis programs for the vax. Nucleic Acids Res. 12,387^395.

[22] Ludwig, W. and Strunk, O. (1997) ARB ^ a software environment forsequence data. Internet address: http://www.biol.chemie.tu-muenchen.de/pub/ARB/documentation/.

[23] Benson, D.A., Boguski, M.S., Lipman, D.J., Ostell, J., Ouellette,B.F.F., Rapp, B.A. and Wheeler, D.L. (1999) GenBank. NucleicAcids Res. 27, 12^17.

[24] Maidak, B.L., Cole, J.R., Parker, C.T., Garrity, G.M., Larsen, N.,Li, B., Lilburn, T.G., McCaughey, M.J., Olsen, G.J., Overbeek, R.,Pramanik, S., Schmidt, T.M., Tiedje, J.M. and Woese, C.R. (1999) Anew version of the RDP (Ribosomal Database Project). NucleicAcids Res. 27, 171^173.

[25] Smith, S.W., Overbeek, R., Woese, C.R., Gilbert, W. and Gillevet,P.M. (1994) The genetic data environment: an expandable GUI formultiple sequence analysis. CABIOS 10, 671^675.

[26] Baumann, L., Baumann, P., Mandel, M. and Allen, R.D. (1972)Taxonomy of aerobic marine eubacteria. J. Bacteriol. 110, 402^429.

[27] Eilers, H., Pernthaler, J., Glockner, F.O. and Amann, R. (2000)

Culturability and In situ abundance of pelagic bacteria from theNorth Sea. Appl. Environ. Microbiol. 66, 3044^3051.

[28] Rappe, M.S. Personal communication.[29] LeBaron, P., Ghiglione, J.-F., Fajon, C., Batailler, N. and Normand,

P. (1998) Phenotypic and genetic diversity within a colony morpho-type. FEMS Microbiol. Lett. 160, 137^143.

[30] Abraham, W.-R., Strompl, C., Meyer, H., Lindholst, S., Moore,E.R.B., Christ, R., Vancanneyt, M., Tindall, B.J., Bennaser, A.,Smit, J. and Tesar, M. (1999) Phylogeny and polyphasic taxonomyof Caulobacter species. Proposal of Maricaulis gen. nov. with Mari-caulis maris (Poindexter) comb. nov. as the type species, and emendeddescription of the genera Brevundimonas and Caulobacter. Int. J. Syst.Bacteriol. 49, 1053^1073.

[31] Uchino, Y., Hirata, A., Yokota, A. and Sugiyama, J. (1998) Reclas-si¢cation of marine Agrobacterium species: proposals of Stappia stel-lulata gen. nov., comb. nov., Stappia aggregata sp. nov., nom. rev.,Ruegeria atlantica gen. nov., comb. nov., Ruegeria gelatinovora comb.nov., Ruegeria algicola comb. nov., and Ahrensia kieliense gen. nov.,sp. nov., nom. rev. J. Gen. Appl. Microbiol. 44, 201^210.

[32] Lafay, B., Ruimy, R. and Rausch De Traubenberg, C. (1995) Roseo-bacter algicola sp. nov., a new marine bacterium isolated from thephycosphere of the toxin-producing dino£agellate Prorocentrum lima.Int. J. Syst. Bacteriol. 45, 290^296.

[33] Ruiz-Ponte, C., Cilia, V., Lambert, C. and Nicolas, J.L. (1998)Roseobacter gallaeciensis sp. nov., a new marine bacterium isolatedfrom rearings and collectors of the scallop Pecten maximus. Int. J.Syst. Bacteriol. 48, 537^542.

[34] Doucette, G.J. and Powell, C.L. (1998) Algal-bacterial interactions:can they determine the PSP-related toxicity of dino£agellates? In:Harmful Algae (Reguera, B., Blanco, J., Fernandez, M.L. and Wyatt,T., Eds.), pp. 406^409. Xunta de Galicia and Intergovernmental Oce-anographic Commission of UNESCO, Gra¢sant, Spain.

[35] Akagi, Y., Taga, N. and Simidu, U. (1977) Isolation and distributionof oligotrophic marine bacteria. Can. J. Microbiol. 23, 981^987.

FEMSEC 1273 9-10-01


characterisation of bacterial communities associated with ......triplicate cell counts using a...

Documents