ORIGINAL PAPER

A comparative study on the phylogenetic diversityof culturable actinobacteria isolated from fivemarine sponge species

Haitao Zhang Æ Wei Zhang Æ Yan Jin ÆMeifang Jin Æ Xingju Yu

Received: 22 April 2007 / Accepted: 6 August 2007 / Published online: 24 August 2007

� Springer Science+Business Media B.V. 2007

Abstract A cultivation-based approach was

employed to compare the culturable actinobacterial

diversity associated with five marine sponge species

(Craniella australiensis, Halichondria rugosa, Reni-

ochalina sp., Sponge sp., and Stelletta tenuis). The

phylogenetic affiliation of the actinobacterial isolates

was assessed by 16S rDNA-RFLP analysis. A total of

181 actinobacterial strains were isolated using five

different culture media (denoted as M1–M5). The type

of medium exhibited significant effects on the number

of actinobacteria recovered, with the highest number

of isolates on M3 (63 isolates) and the lowest on M1

(12 isolates). The genera isolated were also different,

with the recovery of three genera on M2 and M3, and

only a single genus on M1. The number of actinobac-

teria isolated from the five sponge species was

significantly different, with a count of 83, 36, 30, 17,

and 15 isolates from S. tenuis, H. rugosa, Sponge sp.,

Reniochalina sp., and C. australiensis, respectively.

M3 was the best isolation medium for recovery of

actinobacteria from S. tenuis, H. rugosa, and Sponge

sp., while no specific medium preference was

observed for the recovery of actinobacteria from

Reniochalina sp., and C. australiensis. The RFLP

fingerprinting of 16S rDNA genes digested with HhaI

revealed six different patterns, in which 16 represen-

tative 16S rDNAs were fully sequenced. Phylogenetic

analysis indicated that 12 strains belong to the group

Streptomyces, three strains belong to Pseudonocardia,

and one strain belongs to Nocardia. Two strains C14

(from C. australiensis) and N13 (from Sponge sp.)

have only 96.26% and 96.27% similarity to earlier

published sequences, and are therefore potential

candidates for new species. The highest diversity of

three actinobacteria genera was obtained from Sponge

sp., though the number of isolates was low. Two

genera of actinobacteria, Streptomyces, and Pseud-

onocardia, were isolated from both S. tenuis and C.

australiensis. Only the genus of Streptomyces was

isolated from H. rugosa and Reniochalina sp. Sponge

species have been demonstrated here to vary as

sources of culturable actinobacterial diversity, and

the methods for sampling such diversity presented

may be useful for improved sampling of such

diversity.

Keywords Actinobacteria � Nocardia �Pseudonocardia � RFLP � Sponge �Streptomyces � 16S rDNA sequencing

H. Zhang � W. Zhang (&) � Y. Jin � M. Jin � X. Yu

Marine Bioproducts Engineering Group, Dalian Institute

of Chemical Physics, Chinese Academy of Sciences,

Dalian 116023, China

e-mail: WeiZhang@dicp.ac.cn

H. Zhang

Graduate School of the Chinese Academy of Sciences,

Chinese Academy of Sciences, Beijing 100039, China

W. Zhang

Department of Medical Biotechnology, School of

Medicine, Flinders University, Adelaide, SA 5042,

Australia

123

Antonie van Leeuwenhoek (2008) 93:241–248

DOI 10.1007/s10482-007-9196-9

Introduction

Marine sponges (Phylum Porifera) are sessile marine

filter feeders that can filter large volumes of sur-

rounding water through a unique aquiferous system

(Reiswig 1974; De Vos et al. 1991). Many bacteria,

microalgae, and other organic particles in the seawa-

ter can thus be retained and digested by phagocytosis

as foods (Muller et al. 2004). The microorganisms

that are not digested may survive and inhabit the host

sponges. As a result, marine sponges become a rich

reservoir of diverse, highly concentrated marine

bacteria, some of which may not have been cultured

yet. It has been reported that the sponges Aplysina

cavernocola and Ceratoporella nicholsoni harbor a

large number of bacteria that can amount to 38% and

57% of the biomass volume, respectively (Vacelet

1975; Willenz and Hartman 1989). The bacterial

concentration in sponges is estimated to exceed those

present in seawater by about two to four orders of

magnitude (Hentschel et al. 2001). In recent years,

there has been a growing interest in the bacteria

associated with marine sponges as sources of bioac-

tive natural products (Lee et al. 1998; Jayatilake

et al. 1996). This interest has been mainly driven by

the increasing number of bioactive metabolites iso-

lated from sponge-associated bacteria as well as

evidence supporting these bacteria as the real

producers of bioactive metabolites originally isolated

from their host sponges (Schmidt et al. 2000; Stierle

and Stierle 1992; Oclarit et al. 1994).

Among all sponge-associated bacteria, Actinobac-

teria are of particular interest in producing antibiotics

and other therapeutically significant compounds (Ta-

kahashi and Omura 2003). Recent studies showed a

novel and abundant actinobacteria assemblage in the

marine sponge Rhopaloeides odorabile, assessed by

both a culture-independent molecular approach and a

culture-based method (Webster et al. 2001). Several

earlier studies also isolated single strains of actino-

bacteria from marine sponges (Lee et al. 1998;

Imamura et al. 1993). However, our understanding

of the sponge-associated-actinobacteria community is

still inadequate as isolation and exploitation efforts

are just beginning. Further systematic investigations

across more sponge species are required. In the

current study, the questions that we addressed are

whether it is a general feature that many marine

sponges host novel actinobacteria and what the

differences are in the diversity of the actinobacterial

community among sponge species. As a first step to

approach these questions, a cultivation-based method

was used to isolate actinobacteria from five different

sponge species by using five different isolation media

for actinobacteria. These sponge species were col-

lected from the northern and southern parts of China

and the number of actinobacteria isolates and diver-

sity of the culturable actinobacteria community from

these sponge species were directly compared by 16S

rDNA-RFLP analysis.

Material and methods

Sponge collection and isolation of actinobacteria

The marine sponge Reniochalina sp., a newly

recorded species, was collected by hand in the

inter-tidal coast of Yellow Sea around Dalian city,

China. The other four sponges (Halichondria rugosa,

Craniella australiensis, Stelletta tenuis, and Sponge

sp.) from the South China Sea near Sanya City were

collected, maintained on board at 16–20�C and

transported to our laboratory. Sponge specimens

were washed five times in sterile seawater to remove

the loosely attached bacteria, then homogenized in a

mortar, and diluted at 10–1 and 10–2 before plating on

agar plates in triplicate for each dilution. The agar

plates were incubated at 28�C for 2–4 weeks. Five

specialized media were used to isolate sponge-

associated actinobacteria (Webster et al. 2001; Min-

cer et al. 2002) and the medium compositions were:

M1 (10 g of soluble starch, 4 g of yeast extract, 2 g

of peptone, and 1 l of natural seawater); M2 (6 ml of

100% glycerol, 1 g of arginine, 1 g of K2HPO4, 0.5 g

of MgSO4, and 1 l of natural seawater); M3 (2 g of

peptone, 0.1 g of asparagines, 4 g of sodium propi-

onate, 0.5 g of K2HPO4, 0.1 g of MgSO4, 0.01 g of

FeSO4, 5 g of glycerol, 20 g of NaCl, and 1 l of

distilled water); M4 (4 g of yeast extract, 15 g of

soluble starch, 1 g of K2HPO4, 0.5 g of MgSO4, 20 g

of NaCl, and 1 l of distilled water); and M5 (marine

agar 2216 (Difco, USA)). All media contained Difco

Bacto agar (18 g/l) as a solidifying agent and were

each supplemented with a final concentration of

50 lg of K2Cr2O7 ml–1 and 15 lg of nalidixic acid

ml–1. K2Cr2O7 was added to the media to inhibit

fungal growth (Yang et al. 1995) and nalidixic acid to

242 Antonie van Leeuwenhoek (2008) 93:241–248

123

inhibit many fast-growing gram-negative bacteria so

as to allow the isolation of slow-growing actinobac-

teria (Webster et al. 2001). All media were finally

adjusted to pH 7.0.

Strain culture, DNA extraction and PCR

amplification

All the actinobacterial isolates were cultured on

modified Gause’s No.1 agar (20 g of soluble starch,

2 g of NaCl, 1 g of KNO3, 0.01 g of FeSO4, 0.5 g of

K2HPO4, 0.5 g of MgSO4, 18 g of agar, and 1 l of

distilled water). Slow-growing strains were cultured

on TSA agar (Difco, USA). Total DNA was extracted

using the method of Ausubel et al. (1987). For 16S

rDNA gene amplification, genomic DNA was ampli-

fied with bacterial universal primers 8f, 50-GAG AGT

TTG ATC CTG GCT CAG-30, and 1492r, 50-TAC

GGC TAC CTT GTT CTC AG-30 (Webster and Hill

2001). The PCR mixture consisted of 5 ll 10· Buffer

(Mg2+ free), 5 ll 2.5 nM MgCl2, 8 ll dNTP mixture

(2.5 nM each), 1 ll of each primer, 1 ll of template

DNA, and 0.5 ll of LA Taq polymerase (TaKaRa,

China) in a final volume of 50 ll. PCR amplification

parameters were as follows: 95�C and 5 min of initial

melt; 30 cycles of 94�C, 1 min; 55�C, 1 min; and

72�C, 2 min; and a final extension at 72�C for 7 min

(Lee et al. 2003).

RFLP analysis, sequence, and phylogenetic

analysis

The PCR products were digested using the four-cutter

restriction enzyme HhaI (TaKaRa, China) for 2 h and

electrophoresis was performed on a 2% agarose gel

for 3 h at 50 V. The different RFLP patterns were

determined and the isolates were grouped accord-

ingly. The PCR products of 16 typical strains were

purified using an Agarose Gel DNA Fragment

Recovery Kit (TaKaRa, China), and sequenced by

the company TaKaRa. The sequences were edited

using PHYDIT (Chun 1995) and a blast search of the

National Center for Biotechnology Information

(NCBI) was performed to identify the nearest neigh-

bor to the amplified sequence. The sequences were

aligned with actinobacteria 16S rDNA gene data

retrieved from the NCBI website to create a matrix

using CLUSTALW (Thompson et al. 1997). The tree

topologies were evaluated by bootstrap analyses

based on 1,000 replications with PHYLIP (Felsenstein

1993) and phylogenetic trees were inferred using the

neighbor-joining method (Saitou and Nei 1987).

Nucleotide sequence accession numbers

The complete 16S rDNA sequences of 16 representa-

tive strains have been deposited in GenBank database

under the following Accession numbers: AY944250

(N12); AY944251 (N02); AY944252 (N13);

AY944253 (N16); AY944254 (N28); AY944255

(H01); AY944256 (H02); AY944257 (H07);

AY944258 (C07); AY944259 (C06); AY944260

(C14); AY944261 (R02); AY944262 (R03);

AY944263 (S01); AY944264 (S07); AY944265 (S13).

Results

Effect of isolation media on the recoverability of

actinobacteria

A total of 181 actinobacterial isolates were isolated

from the five sponge species using five types of media.

The types of media had a significant effect on the total

number of isolates recovered from five sponge species

(Fig. 1). Medium M3, with the highest inorganic

nutrient content, produced the largest number of

different colony morphotypes (63), followed by M2

M1 M2 M3 M4 M50

10

20

30

40

50

60

70

Num

ber

of a

ctin

obac

teria

l col

ony

mor

phot

ypes

Media Type

NocardiaPseudonocardiaStreptomyces

Fig. 1 Effect of different actinobacterial isolation media on

the total number of morphotypes isolated and the number of

actinobacterial genera isolated from five marine sponge species

Antonie van Leeuwenhoek (2008) 93:241–248 243

123

with 56 isolates and M1 with the smallest number of

isolates (12). The diversity of actinobacteria recov-

ered also varied among different isolation media

(Fig. 1). The largest diversity of actinobacteria was

observed on isolation agar media of M3 and M2 where

three genera of Actinobacteria—Streptomyces,

Pseudonocardia, and Nocardia were isolated; med-

ium M1 yielded the lowest diversity of only one genus

(Streptomyces; Fig. 1).

Numbers of actinobacteria isolated from each

sponge species

The total number of actinobacterial morphotypes

recovered from the five sponge species varied

considerably, with counts of 83, 36, 30, 17, and 15

isolates from S. tenuis, H. rugosa, Sponge sp.,

Reniochalina sp., and C. australiensis, respectively

(Fig. 2). When compared with the other four media,

M3 agar yielded the highest number of cultivable

actinobacteria recovered from H. rugosa. M2 pro-

duced the largest number of isolates from Sponge sp.

A similar number of actinobacteria were isolated on

M3 and M2 from S. tenuis (24 and 25 isolates,

respectively) (Fig. 2). Reniochalina sp., and C.

australiensis showed no specific media preference

among five media tested. Among the five sponge

species, the greatest diversity of actinobacteria iso-

lates was observed for Sponge sp., from which three

actinobacterial genera were isolated; although the

number of isolates (30) was lower in comparison with

that of S. tenuis (83) and H. rugosa (36). Both S.

tenuis and C. australiensis yielded isolates from two

genera (Streptomyces and Pseudonocardia), whereas

H. rugosa and Reniochalina sp., yielded only isolates

from the genus Streptomyces.

RFLP fingerprinting and 16S rDNA sequence

analysis

The restriction endonuclease HhaI was selected for

the digestion of the PCR products of the16S rDNA

for RFLP fingerprinting analysis. Among the 181

strains analyzed, six different RFLP patterns were

visually delineated (Fig. 3). Pattern 1 is represented

by strains H01, R02, N02, and S01; Pattern 2 is

represented by strains C06, H02, N12, and S13;

Pattern 3 is represented by strains C03, N16, and S07;

Pattern 4 is represented by N28 and C14; Pattern 5 is

represented by strains R03 and N13; and only one

strain (H07) exhibited Pattern 6. Among the five

sponge species, actinobacteria from Sponge sp., were

grouped into five patterns; actinobacteria from C.

australiensis, H. rugosa, and S. tenuis were grouped

into three patterns; and only one pattern was found

for actinobacteria from Reniochalina sp. (Fig. 3).

When correlated with the results from 16S rDNA

sequence analysis, it was found that HhaI RFLP

patterns 1, 2, 5, and 6 originated from representatives

of the genus Streptomyces and Pattern 3 originated

from representatives of the genus Pseudonocardia.

However, Pattern 5 originated from representatives of

two genera: Streptomyces (C14) and Nocardia (N28).

Phylogenetic analysis

Based on the RFLP fingerprinting analysis, the16S

rDNA genes of 16 representative strains were fully

sequenced and subjected to phylogenetic analysis.

The sequence results indicate that the dominant

actinobacteria were members of genus Streptomyces,

which were broadly distributed in all of five sponge

species (Fig. 4). Overall 12 strains clustered within

the genus Streptomyces. The phylogenetic analysis

revealed that strains C14, N13, C06, H02, H01, R02,

and S01 were more distantly related to other previ-

ously published Streptomyces. Strains C14 and N13

C. australiensis H. rugosa R. sp. S. tenuis Spon sp. 0

5

10

15

20

25

Num

ber

of a

ctin

bact

eria

l col

ony

orph

otyp

es

Marine Sponge Species

M1 M2 M3 M4 M5

Fig. 2 The number of actinobacterial colony morphotypes

isolated from the five sponge species using the five different

media

244 Antonie van Leeuwenhoek (2008) 93:241–248

123

formed an independent cluster. Their highest 16S

rDNA gene sequence similarities to published

sequences obtained from NCBI/BLAST were 96.26%

and 96.27%, respectively and these two isolates

therefore represent potential candidates for new spe-

cies. Three strains (S07, C03, and N16) from three

sponge species clustered in genus Pseudonocardia

and were only distantly related to their closest

described relatives, which include Pseudonocardia

autotrophica (AJ252824) and Pseudonocardia ant-

arctica (AJ576010). Strain N28 isolated from Sponge

sp., was the only isolate clustered in the genus

Nocardia, with the closest relative being Nocardia

asteroides (AF430026).

Fig. 3 Agarose gel electrophoresis of restriction fragments of

16S rDNA amplification products of actinobacteria associated

with marine sponges digested with restriction enzyme HhaI.

Strain name abbreviation: C, isolates from C. australiensis; H,

isolates from H. rugosa; R, isolates from Reniochalina sp.; S,

isolates from S. tenuis; N, isolates from Sponge sp.

Fig. 4 Neighbor-joining phylogenetic tree from the analysis

of full-length sequences of 16S rDNA genes from actinobac-

teria associated with marine sponges. The numbers at the nodes

are percentages indicating the levels of bootstrap support,

based on a neighbor-joining analysis of 1,000 re-sampled data

sets. Only values of over 50% are shown. The scale bar

represents 0.1 substitutions per nucleotide position

Antonie van Leeuwenhoek (2008) 93:241–248 245

123

Discussion

It has been shown from molecular diversity analysis

of 16S rDNA sequences that Actinobacteria occur

abundantly in marine sponges in several studies

(Hentschel et al. 2001; Imhoff and Stohr 2003;

Montalvo et al. 2005; Webster et al. 2001). It was

found that 30% of the clone sequences obtained from

R. odorabile (Webster et al. 2001) and more than

70% of the clones from specimens of H. panicea

(Imhoff and Stohr 2003) were related to Actinobac-

teria. Given the important role of Actinobacteria in

the production of novel bioactive metabolites, it is

important to understand if the abundance of Actino-

bacteria is a general feature across many sponge

species, as well as the sponge species-specific

associations and diversity. It was notable that all of

the isolates sequenced in our study had 16S rDNA

gene sequences distinct from those of actinobacteria

previously isolated from sponges. The direct com-

parison of the culturable actinobacterial community

in five different sponge species in this study demon-

strated that all sponge species investigated harbor

Actinobacteria, however at vastly different abun-

dance and diversity (Fig. 2). The actinobacterial

abundance is not necessarily related to the species

diversity as indicated for Sponge sp., with the highest

diversity but lowest number of total isolates. It was

observed that the sponge S. tenuis with the highest

number of isolates has a soft surface, is highly porous

and has a dense mesohyl structure, whereas the

sponges C. australiensis and Reniochalina sp., which

have relatively hard body surfaces and less openings

harbored the lowest number of actinobacterial iso-

lates. In fact, over 50% of the dry weight of C.

australiensis was found to contain siliceous spicules

(data not shown). The results suggest that the

abundance and diversity of sponge-associated actino-

bacteria may be greatly affected by different

structures of their aquiferous system. It is not

surprising that Streptomyces, a common actinobacte-

rial genus, is distributed across all five sponge species

but it is notable that some of these isolates did form

independent clusters, indicating a potential sponge-

specific association (Fig. 4).

Cultivation-based methods are always highly

selective due to the choice of media and culture

conditions (Webster et al. 2001; Imhoff and Stohr

2003). This view is illustrated in this study by the fact

that the five isolation media tested exhibited great

differences with regard to the total numbers of

isolates recovered and the diversity of isolates

(Fig. 1). Different isolation media may therefore be

appropriate when examining different sponge species.

Two typical actinobacterial isolation media M2 and

M3 that are widely used for actinobacterial isolation

from the terrestrial environment (Zakharova et al.

2003) showed the best recoverability of isolates for S.

tenuis, Sponge sp., and H. rugosa. However these

were not as effective for the other two sponge species

(Fig. 2). The main carbon and nitrogen sources

contained in M2 and M3 are glycerol, asparagine,

and arginine, which are preferred nutrients of

actinobacteria (Labeda and Shearer 1990). Marine

prokaryotes typically grew better in inorganic media

than in complex organic media (Macleod 1965). This

preference is also valid for the cultivation of the

ubiquitous marine bacterioplankton clade SAR11

(Rappe et al. 2002). Our data may further support

this notion that actinobacteria from marine sponge

are isolated more effectively using defined inorganic

isolation media. Our observation is similar to an

earlier published report, which indicates that different

culture media have diverse effects on the recovery of

actinobacteria isolated from R. odorabile (Webster

et al. 2001). The results also highlight the importance

of testing multiple isolation media and culture

conditions for a better understanding of the culturable

actinobacterial community within sponges.

16S rDNA-RFLP analysis has been widely applied

for the study of the diversity of microbial commu-

nities and for strain identification. It was reported that

actinobacterial strains could be identified at the genus

level using four restriction endonucleases without the

need for sequence analysis (Cook and Meyers 2003).

In our study, the selection of one restriction endonu-

clease HhaI was effective for preliminary 16S rDNA-

RFLP analysis. It is known that actinobacteria belong

to the high ‘‘GC’’ bacteria, where ‘‘GC’’ content

ranges between 60% and 78%. HhaI is a four base-

cutter restriction enzyme, which can specifically

recognize and cut the site ‘‘GCGC’’. In our earlier

study of culturable actinobacterial diversity from the

marine sponge Hymeniacidon perleve it was shown

that HhaI-RFLP analysis of the 16S rDNA gave good

resolution for the identification of the actinobacteria

isolates at the genus level (Zhang et al. 2006a). Based

on the HhaI-RFLP and sequence information of 16S

246 Antonie van Leeuwenhoek (2008) 93:241–248

123

rDNA, a new species was isolated and described

(Zhang et al. 2006b). In this study, the comparison of

the RFLP fingerprints and their 16S rDNA sequences

also could identify the isolated actinobacteria at the

genus level and Streptomyces at sub-genus level.

It should be noted that cultivation-based approaches

are limited since the high selectivity of isolation media

and culture conditions usually allow only a small

fraction of the bacteria present within a sponge

specimen to be isolated (Webster et al. 2001; Imhoff

and Stohr 2003). On the other hand, the use of the 16S

rDNA gene as a phylogenetic marker enables the

determination of the precise phylogenetic position of

sponge bacterial populations independent of their

culturability. Previous studies demonstrated great

differences between the genetically verified diversity

and the cultural spectrum of bacteria from sponges

(Webster et al. 2001; Imhoff and Stohr 2003). In the

case of R. odorabile, the most abundant bacteria

isolated on standard media from 40 sponge specimens

collected from different regions of Great Barrier Reef

was an a-Proteobacterium (Webster and Hill 2001).

However the molecular genetic analysis of the bacteria

diversity indicated that phylum Actinobacteria were

the dominant group in the total bacterial assemblage

(Webster et al. 2001). It is surprising that none of the

previously cultured bacteria from this sponge species

were among those verified by molecular methods.

Therefore, cultivation-based and genetic approaches

are complimentary and should be combined to reveal

the actinobacterial association with marine sponges.

Directed by the information from the culture-indepen-

dent method, actinobacteria could also be cultured

using a suitable isolation approach (Mincer et al.

2005; Rappe et al. 2002). It is necessary to carry out

detailed molecular analyses e.g., using actinobacteria-

specific primers to reveal the true diversity even if

actinobacteria are present as minor components of the

total community. Therefore, it is important to recog-

nize the advantages and limitations of both cultivation-

based and genetic approaches in revealing the actino-

bacterial diversity within marine sponges. In

summary, we found differences in distribution and

diversity of culturable actinobacteria among different

sponge species. The wide distribution of actinobacte-

ria within different sponge species indicates that these

assemblages are a valuable resource for the isolation of

potentially novel actinobacteria for natural product

screening, but that sampling of a diverse range of

sponge species may improve the sampling of sponge

actinobacterial diversity.

Acknowledgements The authors wish to acknowledge

financial support from ‘‘Innovation Fund’’ of Dalian Institute

of Chemical Physics, ‘‘973 Key Basic Science Research

Program of China’’ (2003CB716001), and ‘‘863 Hi-Tech

Research and Development Program of China’’

(2006AA09Z435). Dr. K. Manmadhan’s helps in English

revision is greatly appreciated. We appreciate Professor Jinhe

Li, at Qingdao Institute of Oceanology, Chinese Academy of

Sciences for the sponge identification.

References

Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG,

Smith JA, Struhl K (1987) Current protocols in molecular

biology. Wiley, Cambridge, Massachusetts

Chun J (1995) Computer-assisted classification and identifi-

cation of actinomycetes. Ph.D. Thesis, University of

Newcastle, Newcastle upon Tyne, UK

Cook AE, Meyers PR (2003) Rapid identification of filamen-

tous actinomycetes to the genus level using genus-specific

16S rRNA gene restriction fragment patterns. Int J Syst

Evol Microbiol 53:1907–1915

De Vos L, Rutzler K, Boury-Esnault N, Donadey C, Vacelet J

(1991) Atlas of sponge morphology. Smithsonian Insti-

tution Press, Washington, p 117

Felsenstein J (1993) PHYLIP (Phylogeny inference package),

version 3.5c. Department of Genetics, University of

Washington, Seattle, WA, USA

Hentschel U, Schmidt M, Wagner M, Fieseler L, Gernert C,

Hacker J (2001) Isolation and phylogenetic analysis of

bacteria with antimicrobial activities from the Mediter-

ranean sponges Aplysina aerophoba and Aplysinacavernicola. FEMS Microbiol Ecol 35:305–312

Imamura N, Nishijima M, Adachi K, Sano H (1993) Novel

antimycin antibiotics, urauchimycins A and B, produced

by marine actinomycete. J Antibiot 46:241–246

Imhoff JF, Stohr R (2003) Sponge-associated bacteria: general

overview and special aspects of bacteria association with

Halichondria panacea. In: Muller WEG (ed) Sponge

(Porifera). Springer, Berlin, pp 35–58

Jayatilake GS, Thornton MP, Leonard AC, Grimwade JE,

Baker BJ (1996) Metabolites from an Antarctic sponge-

associated bacterium, Pseudomonas aeruginosa. J Nat

Prod 59:293–296

Labeda D, Shearer M (1990) Isolation of actinomycetes for

biotechnological applications. In: Labeda DP (ed) Isola-

tion of biotechnological organisms from nature. McGraw-

Hill Publishing Company, New York, pp 1–19

Lee HK, Lee DS, Lim J, Kim JS, Jung JH (1998) Topoisom-

erase I inhibitors from the Streptomyces sp. strain KM86-

9B isolated from a marine sponge. Arch Pharm Res

21:729–733

Lee EY, Lee HK, Lee YK, Sim CJ, Lee JH (2003) Diversity of

symbiotic archaeal communities in marine sponges from

Korea. Biomol Eng 20:299–304

Antonie van Leeuwenhoek (2008) 93:241–248 247

123

Macleod RA (1965) The question of existence of specific

marine bacteria. Bacter Rev 29:9–23

Mincer TJ, Jensen PR, Kauffman CA, Fenical W (2002)

Widespread and persistent populations of a major new

marine actinomycete taxon in ocean sediments. Appl

Environ Microbiol 68:5005–5011

Mincer TJ, Fenical W, Jensen PR (2005) Culture-dependent

and culture-independent diversity within the obligate

marine actinomycete genus Salinispora. Appl Environ

Microbiol 71:7019–7028

Montalvo NF, Mohamed NM, Enticknap JJ, Hill RT (2005)

Novel actinobacteria from marine sponges. Antonie Van

Leeuwenhoek 87:29–36

Muller WEG, Grebenjuk VA, Le PG, Schroder HC, Brummer

F, Hentschel U, Muller IM, Breter HJ (2004) Sustainable

production of bioactive compounds by sponges-cell cul-

ture and gene cluster approach: a review. Mar Biotech

6:105–117

Oclarit JM, Okada H, Ohta S, Kaminura K, Yamaoka Y, Iizuka

T, Miyashiro S, Ikegami S (1994) Anti-bacillus substance

in the marine sponge, Hyatella species, produced by an

associated Vibrio species bacterium. Microbios 78:7–16

Rappe MS, Connon SA, Vergin KL, Giovannoni SJ (2002)

Cultivation of the ubiquitous SAR11 marine bacterio-

plankton clade. Nature 418:630–633

Reiswig H (1974) Water transport, respiration and energetics

of three tropical marine sponges. J Exp Mar Biol Ecol

14:231–249

Saitou N, Nei M (1987) The neighbor-joining method: a new

method for reconstructing phylogenetic trees. Mol Biol

Evol 4:406–425

Schmidt EW, Obraztsova AY, Davidson SK, Faulkner DJ,

Haygood MG (2000) Identification of the antifungal

peptide-containing symbiont of the marine sponge Theo-nella swinhoei as a novel d-proteobacterium, ‘‘Candidatus

Entotheonella palauensis’’. Mar Biol 136:969–977

Stierle DB, Stierle AA (1992) Pseudomonic acid derivatives

from a marine bacterium. Experientia 44:1165–1169

Takahashi Y, Omura S (2003) Isolation of new actinomycete

strains for the screening of new bioactive compounds. J

Gen Appl Microbiol 49:141–154

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins

DG (1997). The Clustal X windows interface: flexible

strategies for multiple sequence alignment aided by

quality analysis tools. Nucleic Acid Res 24:4876–4882

Vacelet J (1975) Etude en microscopie electronique de

l’association entre bacteries et spongiaires du genre Ve-rongia (Dictyoceratida). J Microsc Biol Cell 23:271–288

Webster NS, Hill RT (2001) The culturable microbial com-

munity of the Great Barrier Reef sponge Rhopaloeidesodorabile is dominated by a-Proteobacterium. Mar Biol

138:843–851

Webster NS, Wilson KJ, Blackall LL, Hill RT (2001) Phylo-

genetic diversity of bacteria associated with the marine

sponge Rhopaloeides odorabile. Appl Environ Microbiol

67:434–444

Willenz P, Hartman WD (1989) Micromorphology and ultra-

structure of Caribbean sclerosponges I. Ceratoporellanicholsoni and Stromatospongia norae (Ceratoporellidae

Porifera). Mar Biol 103:387–402

Yang Y, Xu L, Li Q, Jiang C (1995) A study on isolationmethods of actinomycetes. Chin J Microbiol 22:88–91

Zakharova OS, Zenova GM, Zvyagintsev DG (2003) Some

approaches to the selective isolation of actinomycetes of

the genus Actinomadura from soil. Microbiology 72:110–

113

Zhang HT, Lee YK, Zhang W, Lee HK (2006a) Culturable

actinobacteria from the marine sponge Hymeniacidonperleve: isolation and phylogenetic diversity by 16S

rRNA gene-RFLP analysis. Antonie Van Leeuwenhoek

90:159–169

Zhang HT, Zheng W, Huang JY, Luo HL, Jin Y, Zhang W, Liu

ZH, Huang Y (2006b) Actinoalloteichus hymeniacidonissp. nov., an actinomycete isolated from the marine sponge

Hymeniacidon perleve. Int J Syst Evol Microbiol

56:2309–2312

248 Antonie van Leeuwenhoek (2008) 93:241–248

123