Widespread Distribution of Poribacteria in Demospongiae

Abstract and Figures

Poribacteria were found in nine sponge species belonging to six orders of Porifera from three oceans. Phylogenetic analysis revealed four distinct poribacterial clades, which contained organisms obtained from several different geographic regions, indicating that the distribution of poribacteria is cosmopolitan. Members of divergent poribacterial clades were also found in the same sponge species in three different sponge genera.
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2009, p. 5695–5699 Vol. 75, No. 17
0099-2240/09/$08.000 doi:10.1128/AEM.00035-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Widespread Distribution of Poribacteria in Demospongiae
Feras F. Lafi,
John A. Fuerst,
* Lars Fieseler,
† Cecilia Engels,
Winnie Wei Ling Goh,
and Ute Hentschel
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia,
Research Center for Infectious Diseases, University of Wu¨rzburg, Ro¨ntgenring 11, D-97070 Wu¨rzburg, Germany
Received 7 January 2009/Accepted 21 June 2009
Poribacteria were found in nine sponge species belonging to six orders of Porifera from three oceans.
Phylogenetic analysis revealed four distinct poribacterial clades, which contained organisms obtained from
several different geographic regions, indicating that the distribution of poribacteria is cosmopolitan. Members
of divergent poribacterial clades were also found in the same sponge species in three different sponge genera.
Recently, a novel bacterial phylum, termed “Poribacteria,”
was discovered, and members of this phylum have been found
exclusively in sponges (2). Phylogenetic analyses of 16S rRNA
genes indicated that poribacteria are evolutionarily deeply
branching organisms and related to a superphylum composed
of Planctomycetes,Verrucomicrobia, and Chlamydia (11). Pori-
bacterial 16S rRNA genes contain 13 of 15 planctomycete
signature nucleotides, but a level of sequence divergence of
more than 25% compared to any other bacterial phylum, in-
cluding the Planctomycetes, justifies the status of this taxon as
an independent phylum. A consistent treeing pattern is difficult
to resolve in comparative phylogenetic sequence analyses,
making the poribacteria an unusual line of phylogenetic de-
scent. In addition to their divergent status as a separate phylum
on the basis of the 16S rRNA sequence, poribacteria are also
divergent because they may have a compartmentalized cell
structure, a cell plan they share only with members of the phyla
Planctomycetes and Verrucomicrobia (2). They are also of in-
terest for understanding the potential contribution of obligate
sponge-associated bacteria to the sponges harboring them and
as an example of a yet-to-be-cultured group of bacteria asso-
ciated with invertebrate tissue apparently exclusively but for
unknown reasons. This study aimed to further explore the
presence and diversity of poribacteria in different marine
demosponge genera using samples from around the world.
The Mediterranean sponges were collected by scuba divers
offshore at Banyuls sur Mer, France (42°29N, 03°08E). The
Caribbean sponges were collected offshore at Little San Sal-
vador Island, Bahamas (24°32N, 75°55W). The eastern Pa-
cific sponge Aplysina fistularis was collected offshore at San
Diego, CA (32°51N, 117°15W). The western Pacific sponge
Theonella swinhoei was collected offshore at Palau (07°23N,
134°38E). All non-Great Barrier Reef (non-GBR) sponges
were collected between May and July 2000, and once individ-
ual sponge specimens were brought to the surface, they were
frozen in liquid nitrogen on board ship and stored at 80°C
until microbiological processing (9). The GBR marine sponges
were collected off Heron Island Research Station (23°27S,
151°5E) in April 2002 (5). Pseudoceratina clavata was col-
lected by scuba divers at a depth of 14 m, and Rhabdastrella
globostellata was collected at a depth of ca. 0.5 m after a reef
walk consisting of a few hundred meters. The samples were
immediately placed in plastic bags and brought to Heron Is-
land Research Station, where they were stored at 80°C until
processing. Sponge DNA was extracted as described previously
(2, 5).
Total sponge-derived genomic DNA was screened by PCR
for the presence of poribacteria using a 16S rRNA gene primer
set. Poribacterial 16S rRNA genes were amplified by employ-
ing a pair of Poribacteria-specific primers, POR389f (5-ACG
ATG CGA CGC CGC GTG-3) and POR1130r (5-GGC
TCG TCA CCA GCG GTC-3) (2). The poribacterial PCR
products that were ca. 740 bp long derived from one sponge
individual were cloned into the pGEM-T Easy vector (Pro-
mega, Madison, WI). Clone inserts were digested with restric-
tion endonucleases MspI and HaeIII (New England Biolabs,
Inc., United States), characterized to obtain restriction profiles
and unique profiles, and sequenced. The compiled partial 16S
rRNA gene sequences were then analyzed using BLASTN to
select the most closely related poribacterial reference se-
The sequences exhibiting levels of similarity of less than 97%
were used for further analysis. Poribacterial 16S rRNA gene
sequences were aligned using the ARB software package (7).
The resulting alignment was imported into PAUP (10) and
analyzed by using distance, maximum parsimony, and maxi-
mum likelihood algorithms together with bootstrap resam-
plings (3,000, 3,000, and 200 resamplings, respectively), and the
resulting bootstrap values were applied to nodes on the ARB
neighbor-joining tree. Signature sequences were detected us-
ing the ARB software package. A signature sequence is de-
fined here as a short sequence that is present in a group of
poribacterial sequences in a phylogenetic clade but is not
found in any other clade in the poribacterial tree.
Analysis of the 16S rRNA gene clone library sequences
generated from sponge tissues revealed the presence of pori-
bacteria in sponge individuals belonging to the orders Verongida,
Astrophorida, Dictyoceratida, Haplosclerida, Lithistida, and
Homosclerophorida, while poribacteria could not be detected
* Corresponding author. Mailing address: School of Chemistry and
Molecular Biosciences, The University of Queensland, Brisbane,
Queensland 4072, Australia. Phone: (49) 931 312581. Fax: (49) 931
312578. E-mail:
Present address: Institute for Food Science and Nutrition, ETH
Zurich, Schmelzbergstrasse 7, CH-8092 Zurich, Switzerland.
Published ahead of print on 26 June 2009.
in sponges belonging to the orders Hadromerida and Agela-
sida. In the order Halichondrida, poribacteria were detected in
Xestospongia muta but not in Haliclona sp. Altogether, nine
sponge species were added to the list of Poribacteria-containing
sponges (Table 1). Three distinct clades were observed that
were clearly supported by bootstrap values greater than 75 with
every tree-building algorithm applied (Fig. 1), and one clade
(clade I) was supported by bootstrap values of 64, 98, and 71 in
distance, maximum parsimony, and maximum likelihood trees,
respectively. Similarity calculations using approximately
740-bp amplified poribacterial 16S rRNA gene fragments and
other poribacterial sequences from the NCBI database showed
TABLE 1. Distribution of poribacteria in different demosponge orders
Sponge species or seawater Order Geographic location
Presence of
Aplysina aerophoba Verongida MED 2
Aplysina lacunosa Verongida BAH 2
Aplysina fistularis Verongida EPAC or BAH 2
Aplysina insularis Verongida BAH 2
Verongula gigantea Verongida BAH 2
Smenospongia aurea Dictyoceratida BAH 2
Aplysina cauliformis Verongida BAH This study
Aplysina archeri Verongida BAH This study
Aplysina cavernicola Verongida MED This study
Pseudoceratina clavata Verongida WPAC This study
Rhabdastrella globostellata Astrophorida WPAC This study
Ircinia sp. Dictyoceratida BAH This study
Xestospongia muta Haplosclerida BAH This study
Theonella swinhoei Lithistida EPAC This study
Plakortis sp. Homosclerophorida BAH This study
Chondrilla nucula Hadromerida BAH 2
Agelas wiedenmayeri Agelasida BAH 2
Agelas cerebrum Agelasida BAH This study
Axinella polypoides Halichondrida MED This study
Ptilocaulis sp. Halichondrida BAH 2
Dysidea avara Dictyoceratida MED This study
Haliclona sp. Haplosclerida MED This study
Ectyoplasia ferox Poecilosclerida BAH 2
Seawater NA
MED This study
MED, Mediterranean Sea; BAH, Bahamas; WPAC, western Pacific Ocean; EPAC, eastern Pacific Ocean.
The presence of poribacteria was evaluated by sequencing and phylogenetic analysis of amplified PCR products. , present; , absent.
NA, not applicable.
FIG. 1. Neighbor-joining phylogenetic tree for poribacterial clones based on Poribacteria-specific PCR products (740 bp) of the 16S rRNA gene,
showing relationships of poribacterial clones from different global regions. The poribacterial clones on the right are additional clones belonging
to the same clades as strains in the tree at the same level. Bootstrap confidence values of 75% for distance, maximum parsimony, and maximum
likelihood algorithm analyses are indicated by filled circles at nodes, and open circles indicate unsupported nodes. Prefixes for clones: A, Aplysina
aerophoba;C,Aplysina cavernicola;F,Aplysina fistularis;L,Aplysina lacunosa;S,Ircinia sp.; P, Plakortis sp.; PC, Pseudoceratina clavata; RG,
Rhabdastrella globostellata;T,Theonella swinhoei;X,Xestospongia muta. Scale bar 0.1 nucleotide substitution per site.
that the dissimilarity between clades was consistent with their
separation in phylogenetic trees. For example, the levels of
dissimilarity between members of clade I and clade II were 3 to
8%, while the levels of dissimilarity between members of clades
I and III and between members of clades I and IV were 10 to
14% and 11 to 15%, respectively.
Within each clade in the phylum Poribacteria, there were
higher similarity values, including 94 to 100% among members
of clade I, 94 to 99% among members of clade II, 96 to 99%
among members of clade III, and 96 to 99% among members
of clade IV. When members of the the phylum Poribacteria
were compared to members of the Planctomycetes (Fig. 1), the
16S rRNA genes exhibited levels of sequence dissimilarity of
up to 38%, consistent with the conclusion of Fieseler et al.
concerning the separate phylum level status of poribacteria
based on a similarity value of 75%. A phylogenetic correla-
tion between sponge phylogeny and poribacterial phylogeny is
not evident, since, for example, clones from A.fistularis and
Aplysina aerophoba occurred in both clade I and clade II and
one clone from A. aerophoba also occurred in clade III, while
clones from P.clavata and R.globostellata occurred in clades I,
II, and III but not in clade IV. Clades I and II included
poribacterial clones derived from all sponge species occurring
in all of the widely separated geographic regions examined in
this study (Fig. 2). Clade III represented poribacterial clones
derived from sponge species obtained in the eastern Pacific
region, GBR, and the Bahamas but not in the Mediterranean
region. The majority of poribacterial clones in clade IV were
derived from sponge species obtained in the Bahamas, and one
clade IV clone was obtained from a sponge species collected in
the Mediterranean region.
Poribacterial clones from different sponges from widely sep-
arated marine habitats belonged to at least four major clades
with similarities ranging from 94 and 96%. For clade III (Fig.
1), we detected a signature sequence characteristic of poribac-
terial clones from the GBR sponges R.globostellata and P.
clavata. This signature sequence (CCA GTT AGC TTG ACG
GTA) (Table 2) at E. coli positions 469 to 487 targeted 10
sequences, 5 of which were from GBR marine sponges gener-
ated in this study (clones RG68, RG112, RG105, PC96, and
PC8). Another five poribacterial sequences were detected in an
unpublished study investigating the microbial diversity in GBR
sponges. This signature sequence indicates a specific geo-
graphic presence of poribacteria belonging to clade IV in the
GBR region. In addition, a sequence (GAG TGT GAA ATG
GCT TGG at E. coli positions 599 to 617) characteristic of
clade IV was found in 11 sequences derived from sponges from
the Bahamas and one sequence (A7) from a Mediterranean
Based on the data presented here, Poribacteria appears to be
a bacterial phylum that is specifically found in several demo-
sponge genera of the phylum Porifera (Table 1). To our knowl-
edge, this is the only case of a bacterial phylum specifically
associated with a marine invertebrate phylum. Certain phylum
members appear to be widely distributed among sponges be-
longing to different species and in different geographic regions,
forming sponge-specific lineages (3), but these are individual
species level or at most genus level clades in a subdivision of a
phylum rather than in a whole phylum.
PCR analyses of seawater samples collected in this study
(Table 1) and searches using nucleotide sequence databases of
seawater metagenomes were negative for poribacteria. This is
consistent with the concept that Poribacteria is a sponge-spe-
cific phylum. Within the sponges poribacteria are distributed
FIG. 2. Neighbor-joining phylogenetic tree for poribacterial clones based on Poribacteria-specific PCR products (740 bp) of the 16S rRNA gene,
showing the internal relationships of and occurrence of clade I members in distinct sponge species representing cosmopolitan geographic regions.
For an explanation of the colors, see Fig. 1. Bootstrap confidence values of 75% for distance, maximum parsimony, and maximum likelihood
algorithm analyses are indicated by filled circles at nodes, and open circles indicate unsupported nodes. Prefixes for clones: A, Aplysina aerophoba;
C, Aplysina cavernicola;F,Aplysina fistularis;L,Aplysina lacunosa;S,Ircinia sp.; P, Plakortis sp.; PC, Pseudoceratina clavata; RG, Rhabdastrella
globostellata;T,Theonella swinhoei;X,Xestospongia muta. Scale bar 0.1 nucleotide substitution per site. Clones PC15, L8, T6, C2, P3, S2, and
X1 were removed from this analysis to allow better branch resolution.
among members of distinct demosponge orders that occur in
various geographic locations, indicating that there is wide dis-
tribution of poribacteria among marine demosponges. Very
similar 16S rRNA clone sequences that cluster in clade I were
found in sponges from all geographic regions sampled in this
study, including locations in the Northern and Southern hemi-
spheres (Fig. 2). Similarly, clade II contains poribacterial
clones from the Mediterranean Aplysina species and from
GBR Pseudoceratina and Rhabdastrella species. This appears
to contrast, albeit at a lower level of resolution, with results
suggesting that bacterial populations are endemic in different
geographic regions, e.g., with the findings that marine bacte-
rioplankton communities include few cosmopolitan opera-
tional taxonomic units (8), that fluorescent Pseudomonas ge-
notypes from soil are endemic at different geographic sites (1),
and that hyperthermophilic Sulfolobus archaea from different
geothermal areas are genetically divergent (12). Judging the
endemicity of populations in different geographic regions may
depend on the taxonomic scale used to distinguish populations
(1). In this study we provide evidence that at least some clades
may be relatively characteristic of particular regions, e.g., GBR
clade III (Table 2). It is remarkable that in the case of the
sponge species R.globostellata and P.clavata from a single
geographic region (GBR), the microbial communities include
representatives of distantly related poribacterial clades II and
III, whose sequences exhibit levels of dissimilarity ranging
from 10 to 13%. In another case poribacteria belonging to
clades I, II, and IV were found in a single host, A.aerophoba,
from the Mediterranean. Thus, members of widely divergent
poribacterial clades occur in the same specimen in sponges in
widely separated geographic regions in the world’s oceans.
Three different sponge species belonging to three different
genera exhibit this phenomenon.
The morphology and life strategy of sponges have remained
unchanged for the past 580 million years, as judged by the
dramatic similarity of the morphologies of Precambrian fossils
to the morphologies of recent sponges (6). Adaptation of the
poribacteria to this niche might have taken place early in evo-
lution before the various sponge orders separated from each
other. It seems likely that poribacteria diverged from other
bacterial phyla long before evolution of the metazoans as part
of the fan-like radiation by which all bacterial phyla appear to
have arisen (4). This bacterial radiation may have resulted in
the divergence of the clades that we have observed for the
poribacteria, but there is no indication of cospeciation between
host sponges and the poribacteria.
In summary, poribacteria exhibit considerable diversity and
are classified into four phylogenetic clades. Poribacteria seem
to be widely distributed among many different marine demo-
sponge genera, and further studies are needed to explain the
nature of the poribacterium-sponge interaction.
Nucleotide sequence accession numbers. 16S rRNA gene
sequences obtained in this study have been deposited in the
EMBL/GenBank/DDBJ databases under accession numbers
EU071627 to EU071680.
We thank the marine operations personnel at the Institute Rudjer
Boscovic (Rovinj, Croatia) for excellent support, as well as the captain
and crew of the R/V Seaward Johnson (HBOI, Florida) for their help
during sponge collection. We thank Mary Garson and John Hooper for
assistance with collection and identification of GBR sponges from
Financial support was provided by a Deustche Forschungsgemein-
schaft SFB567 grant (TP C3) to U.H. and by grants from the Austra-
lian Research Council and the University of Queensland to J.A.F.
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TABLE 2. Poribacterial signature sequences for clades III and IV, including a GBR-specific signature sequence (pori_SSIII) and a signature
sequence specific to 11 of 12 sequences from the Bahamas (pori_SSIV)
sequence Name Full name
E. coli
position Sequence
pori_SSIII Pla101P Pla101P* 469 GGUGAUAAG-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CCAUAGUA
Pla131P Pla131P* 469 GGUGAUAAG-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CCAUAGUA
Pla134P Pla134P* 469 GGUGAUAAG-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CCAUAGUA
Pla50P Pla50P* 469 GGUGAUAAG-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CCAUAGUA
Pla82P Pla82P* 469 GGUGAUAAG-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CCAUAGUA
PO68 Pori clone RGPo68 469 GGUGAUAAG-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-GAGAAAAG
PO112 Pori clone RGPo112 469 GGUGAUAAU-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CCAUAGUA
PO105 Pori clone RGPo105 469 GGUGAUAAG-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CCAUAGUA
PO96 Uncultured Pori clone 469 GGUGAUAAG-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CCAUAGUA
PCPO8 Pori clone PCPo8 469 GGUGAUAAG-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CCAUAGUA
pori_SSIV AY485286 Uncultured Pori clone 599 ACAUUAGUC-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CUCAACCA
AY485285 Uncultured Pori clone 599 ACAUNAGUC-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CUCAACNA
AY485284 Uncultured Pori clone 599 ACAUUAGUC-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CUCAACCA
AY485281 Uncultured Pori bacterium 599 ACAUUAGUC-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CUCAACCA
A7 A7 599 AUAUUAGUC-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CUCAACCA
F2 F2 599 ACAUAAGUC-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CUCAACCA
L16 L16 599 ACAUUAGUC-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CUCAACCA
P20 P20 599 ACAUAAGUC-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CUCAACCA
P38 P38 599 ACAUUAGUC-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CUCAACCA
S6 S6 599 AUAUUAGUC-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CUCAACCA
S10 S10 599 AUAUUAGUC-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CUCAACCA
X18 X18 599 ACAUUAGUC-⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽⫽-CUCAACCA
Asterisks indicate poribacterial clones derived from the GBR sponge R. globostellata in a separate study.
The internal sequence (indicated by equals signs) of each pori_SSIII clone is CCAGUUAGCUUGACGGUA, and that of each pori_SSIV clone is GAGUGU
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Supplementary resources (54)

... Other factors, such as collection site or depth, could not explain the observed trend. Similar incongruence of symbiont and host phylogeny was observed for the Entoporibacteria (34 homologous genes used to estimate synonymous substitution rates) ( Fig. 4C and D), in agreement with previous phylogenetic studies (34,36,37). This would suggest that these sponges likely acquired a free-living Tethybacterales common ancestor at different time points throughout their evolution and that the same is true for the Entoporibacteria. ...
Full-text available
The fossil record indicates that the earliest evidence of extant marine sponges (phylum Porifera) existed during the Cambrian explosion and that their symbiosis with microbes may have begun in their extinct ancestors during the Precambrian period. Many symbionts have adapted to their sponge host, where they perform specific, specialized functions. There are also widely distributed bacterial taxa such as Poribacteria, SAUL, and Tethybacterales that are found in a broad range of invertebrate hosts. Here, we added 11 new genomes to the Tethybacterales order, identified a novel family, and show that functional potential differs between the three Tethybacterales families. We compare the Tethybacterales with the well-characterized Entoporibacteria and show that these symbionts appear to preferentially associate with low-microbial abundance (LMA) and high-microbial abundance (HMA) sponges, respectively. Within these sponges, we show that these symbionts likely perform distinct functions and may have undergone multiple association events, rather than a single association event followed by coevolution. IMPORTANCE Marine sponges often form symbiotic relationships with bacteria that fulfil a specific need within the sponge holobiont, and these symbionts are often conserved within a narrow range of related taxa. To date, there exist only three known bacterial taxa (Entoporibacteria, SAUL, and Tethybacterales) that are globally distributed and found in a broad range of sponge hosts, and little is known about the latter two. We show that the functional potential of broad-host range symbionts is conserved at a family level and that these symbionts have been acquired several times over evolutionary history. Finally, it appears that the Entoporibacteria are associated primarily with high-microbial abundance sponges, while the Tethybacterales associate with low-microbial abundance sponges.
... In fact, sponge-associated microorganisms are of great interest because of their potential role in producing several compounds (Montalvo and Hill 2011;Moitinho-Silva et al. 2017). Several studies of marine sponges-related bacteria have identified novel bacterial groups, such as the candidate phylum Poribacteria (Lafi et al. 2009). Moreover, bacteria also play a significant role in eliminating polycyclic aromatic hydrocarbons (PAHs) from contaminated habitats but, the potential use of marine sponges with symbiont bacteria is still unexplored (Marzuki 2018). ...
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Marzuki I, Kamaruddin M, Ahmad R. 2021. Identification of marine sponges-symbiotic bacteria and their application in degrading polycyclic aromatic hydrocarbons. Biodiversitas 22: 1481-1488. Diverse and abundant microbial species that occupy marine sponges may make important contributions to host metabolism. Sponges are filter feeders and devour microorganisms from the seawater around them. Each microbe that endures the sponges’ digestive and immune responses are related symbiotically. Marine sponges symbiont bacteria can comprise as much as 40% of sponge tissue volume, and these are known to exhibit a great potential on polycyclic aromatic hydrocarbons (PAHs) degradation. However, the potential use of marine sponges symbiont bacteria is unexplored. Therefore, we designed and conducted a study to identify bacterial isolates obtained from sponges. For this, we collected sponges samples (Hyrtios erectus, Clathria (Thalysias) reinwardti), Niphates sp., and Callyspongia sp.) from the Spermonde islands in Indonesia. We successfully found eight bacterial isolates from four sponges, as molecular identification based on 16S rRNA approach revealed bacterial isolates of SpAB1, SpAB2, SpBB1, SpDB1, and SpDB2 from three sponges (Hyrtios erectus, Clathria (Thalysias) reinwardti), Niphates sp.). Interestingly, these were closely related to Pseudomonas, and a bacterial isolate from Callyspongia sp. (SpCB1) showed similarity to Bacillus. Bacillus and Pseudomonas bacteria isolated from hydrocarbon-contaminated sponges exhibited degradation of naphthalene and pyrene PAHs.
... Culture-independent, as well as culture-dependent studies revealed that sponges have diverse bacterial populations. Phylogenetic studies clearly showed more than taxa belonging to thirty different bacterial phyla (Schleifer 2004;Webster et al. 2008;Sipkema et al. 2009), including the candidate phylum 'Poribacteria' (exclusively found within sponges; Fieseler et al. 2004;Lafi et al. 2009) are associated with sponges. ...
Five sponge specimens belonging to the genera Spongilla and Ciocalypta were collected from Little Rann of Kutch (in Gujarat, India) and analysed for associated microbiomes. Critical analysis was done with respect to members of the phylum Planctomycetes, using two different strategies; 1. Culture-independent metagenomic approach and 2. culture-dependent anaerobic enrichment for anammox-planctomycetes. The 16S rRNA gene (V1-V3 region) amplicon metagenome analysis revealed significant divergence in bacterial diversity, including Planctomycetes among the sponges analysed. Community metagenomics revealed a total of 376 Operational Taxonomic Units (OTUs) belonging to 41 different phyla. OTUs belonging to Proteobacteria was the most abundant (38%) among the sponge analysed. The KEGG annotation predicted a total of 6909 KEGG orthologs (KOs); most of the KOs are associated with membrane transport, xenobiotic degradation, production of secondary metabolites, amino acid metabolism and carbohydrate metabolism. In the culture-dependent study, FISH analysis confirmed the association of anammox-planctomycetes with sponges. Partial 16S rRNA gene sequences of two planctomycetes (JC545, JC543) were cladding with those of uncultured Phycisphaerae class. The other three putative anammox bacteria (JC541, JC542, JC544) formed a different clade with "Candidatus Brocadia anammoxidans". These three putative bacteria believably represent new species/genus related to "Candidatus Brocadia".
... This becomes apparent by striking examples such as Pseudoalteromonas luteoviolacea's contractile injection system injecting tubeworms metamorphosis factors into host cells (Ericson et al., 2019, Shikuma et al., 2014, or even basic mechanisms of sporulation (Khanna et al., 2019), which, functionally, would have been hardly understood by genomic evidence alone. CHAPTER 3 provided the first clear evidence that supports the long standing hypothesis of cell compartmentation (Fieseler et al., 2004 in a lineage of sponge endemic Poribacteria (Podell et al., 2019, Steinert et al., 2018a, Lafi et al., 2009. This revealed that the poribacterial cell structure is more complex than previously thought. ...
Holobionts result from intimate associations of eukaryotic hosts and microbes and are now widely accepted as ubiquitous and important elements of nature. Marine sponge holobionts combine simple morphology and complex microbiology whilst diverging early in the animal kingdom. As filter feeders, sponges feed on planktonic bacteria, but also harbour stable species-specific microbial consortia. This interaction with bacteria renders sponges to exciting systems to study basal determinants of animal-microbe symbioses. While inventories of symbiont taxa and gene functions continue to grow, we still know little about the symbiont physiology, cellular interactions and metabolic currencies within sponges. This limits our mechanistic understanding of holobiont stability and function. Therefore, this PhD thesis set out to study the questions of what individual symbionts actually do and how they interact. The first part of this thesis focuses on the cell physiology of cosmopolitan sponge symbionts. For the first time, I characterised the ultrastructure of dominant sponge symbiont clades within sponge tissue by establishing fluorescence in situ hybridization-correlative light and electron microscopy (FISH-CLEM). In combination with genome-centred metatranscriptomics, this approach revealed structural adaptations of symbionts to process complex holobiont-derived nutrients (i.e., bacterial microcompartments and bipolar storage polymers). Next, we unravelled complementary symbiont physiologies and cell co-localisation indicating vivid symbiont-symbiont metabolic interactions within the holobiont. This suggests strategies of nutritional resource partitioning and syntrophy to dominate over spatial segregation to avoid competitive exclusion- a mechanistic framework to sustain high microbial diversity. By combining stable isotope pulse-chase experiments with metabolic imaging, we demonstrated that symbionts can account for up to 60 % of the heterotrophic carbon and nitrogen assimilation in sponges. Thus, sponge symbiont action determines sponge-driven biochemical cycles in marine ecosystems. Finally, I explored the role of phages in the sponge holobiont focussing on tripartie phage-microbe-host interplay. Sponges appeared as rich reservoirs of novel viral diversity with 491 previously unidentified genus-level viral clades. Further, sponges harboured highly individual, yet species-specific viral communities. Importantly, I discovered that phages, termed “Ankyphages”, abundantly encode ankyrin proteins. Such “Ankyphages” I found to be widespread in host-associated environments, including humans. Using macrophage infection assays I showed that phage ankyrins aid bacteria in eukaryote immune evasion by downregulating eukaryotic antibacterial immunity. Thus, I identified a potentially widespread mechanism of tripartite phage-prokaryote-host interplay where phages foster animal-microbe symbioses. Altogether, I draw three main conclusions: The sponge holobiont is a metabolically intertwined ecosystem, with symbiont action impacting the environment, and tripartite phage-prokaryote-eukaryote interplay fostering symbiosis.
... Chemoautotrophs such as nitrifying archaea, bacteria and mixotrophic Poribacteria have a cosmopolitan distribution in sponges and the genomic repertoire for chemotrophy (Preston et al. 1996;Hallam et al. 2006;Taylor et al. 2007;Lafi et al. 2009;Siegl et al. 2011;Simister et al. 2012;Cardoso et al. 2013;Li et al. 2014). They fix inorganic carbon by obtaining the energy from the oxidation of reduced inorganic compounds e.g. ...
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Many cold-water sponges harbour microorganisms of which the role in the sponge host remains enigmatic. Here, we show a transfer of fixed inorganic carbon by sponge-associated microbes to its host, the cold-water coral encrusting sponge Hymedesmia (Stylopus) coriacea. Sponge were collected at approx. 100 m depth and incubated for 1.5–2.5 days with ¹³C labelled dissolved inorganic carbon (DIC) as tracer. Total DIC fixation rates ranged from 0.03–0.11 mmol C × mmol Csponge × d⁻¹. ¹³C-tracer was recovered in bacterial-specific (i.e. short and branched) and sponge-specific (very long-chained) phospholipid-derived fatty acids (PLFA's), but was not incorporated into archaeal lipids. ¹³C-incorporation in biomarkers such as C16:1w7c and C18:1w7c indicated that nitrifying and/or sulphur-oxidizing bacteria (chemoautotrophs) were likely active in the sponge. Trophic transfer of microbially-fixed carbon to the sponge host was confirmed by recovery of label in very long chain fatty acids (VLCFA's) including C26:2 and C26:3. Tracer accumulation into several VLCFA's continued after removal of ¹³C-DIC, while tracer in most bacteria-specific PLFA's declined, indicating a transfer and elongation of bacterial-specific PLFA's to sponge-specific PLFA's. This implies that PLFA precursors released from chemo- as well as heterotrophic microbes in sponges contributed to the synthesis of VLCFA's, identifying sponge-associated bacteria as symbionts of the sponge.
Host-specific microbial communities thrive within sponge tissues and this association between sponge and associated microbiota may be driven by the organohalogen chemistry of the sponge animal. Several sponge species produce diverse organobromine secondary metabolites (e.g. brominated phenolics, indoles, and pyrroles) that may function as a chemical defense against microbial fouling, infection or predation. In this study, anaerobic cultures prepared from marine sponges were amended with 2,6-dibromophenol as the electron acceptor and short chain organic acids as electron donors. We observed reductive dehalogenation from diverse sponge species collected at disparate temperate and tropical waters suggesting that biogenic organohalides appear to enrich for populations of dehalogenating microorganisms in the sponge animal. Further enrichment by successive transfers with 2,6-dibromophenol as the sole electron acceptor demonstrated the presence of dehalogenating bacteria in over 20 sponge species collected from temperate and tropical ecoregions in the Atlantic and Pacific Oceans and the Mediterranean Sea. The enriched dehalogenating strains were closely related to Desulfoluna spongiiphila and Desulfoluna butyratoxydans, suggesting a cosmopolitan association between Desulfoluna spp. and various marine sponges. In vivo reductive dehalogenation in intact sponges was also demonstrated. Organobromide-rich sponges may thus provide a specialized habitat for organohalide-respiring microbes and D. spongiiphila and/or its close relatives are responsible for reductive dehalogenation in geographically widely distributed sponge species.
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Marine sponges host a wide diversity of microorganisms, which have versatile modes of carbon and energy metabolism. In this study we describe the major lithoheterotrophic and autotrophic processes in 21 microbial sponge-associated phyla using novel and existing genomic and transcriptomic datasets. We show that the main microbial carbon fixation pathways in sponges are the Calvin–Benson–Bassham cycle (energized by light in Cyanobacteria, by sulfur compounds in two orders of Gammaproteobacteria, and by a wide range of compounds in filamentous Tectomicrobia), the reductive tricarboxylic acid cycle (used by Nitrospirota), and the 3-hydroxypropionate/4-hydroxybutyrate cycle (active in Thaumarchaeota). Further, we observed that some sponge symbionts, in particular Acidobacteria, are capable of assimilating carbon through anaplerotic processes. The lithoheterotrophic lifestyle was widespread and CO oxidation is the main energy source for sponge lithoheterotrophs. We also suggest that the molybdenum-binding subunit of dehydrogenase (encoded by coxL) likely evolved to benefit also organoheterotrophs that utilize various organic substrates. Genomic potential does not necessarily inform on actual contribution of autotrophs to light and dark carbon budgets. Radioisotope assays highlight variability in the relative contributions of photo- and chemoautotrophs to the total carbon pool across different sponge species, emphasizing the importance of validating genomic potential with physiology experimentation.
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Ten bromotyrosine alkaloids( 1 ‐ 10 ) were isolated and characterised from the marine sponge Aplysinella rhax ( de Laubenfels 1954) collected from the Fiji Islands, which included one new bromotyrosine analogue, psammaplin P ( 6 ) and two other analogues, psammaplins O ( 5 ) and 4‐bromo‐6‐carbomethoxy salicylic acid ( 7 ), which have not been previously reported from natural sources. HRESIMS, 1D and 2D NMR spectroscopic methods were used in the elucidation of the compounds. Bisaprasin, a biphenylic dimer of psammaplin A, showed moderate activity with IC 50 at 19+/‐ 5 and 29+/‐ 6 μM against Trypanzoma cruzi Tulahuen C4, and the lethal human malaria species Plasmodium falciparum clone 3D7, respectively, while psammaplins A ( 1 ) and D ( 4 ) exhibited low activity against both parasites. This is the first report of the antimalarial and antitrypanosomal activity of the psammaplin‐type compounds. Additionally, the biosynthesis hypotheses of the three natural products ( 5 , 6 and 7 ) were proposed.
The ocean is the largest habitat on our planet for microbes. These microorganisms play a key role in global biogeochemical cycles. Still, we are missing more detail on their phylogenetic, genomic, and metabolic diversity. Microbes play a key role in the sponge and coral biology. Sponges and corals can no longer be considered as autonomous entities but rather as holobionts. Microbes contribute to the nutrition, defense, immunity, and development of the host. Associated microbes can comprise as much as half of their tissue volume, with densities in excess of a billion cells of the sponge tissue, several orders of magnitude higher than those typical for seawater. Each of the three domains of life, i.e., Bacteria, Archaea, and Eukarya (single-celled eukaryotes: fungi and microalgae), is now known to reside within sponges and corals. This chapter focuses on domains of life that are present in sponges and corals and gives an overview of their biodiversity and significance: (1) microbes in sponge and (2) microbes in corals.
Lack of sufficient pure natural compounds hinders further drug developments. The optimization of fermentation conditions is essential to enhance the yield of metabolites. Microbial genome analysis reveals the presence of a large number of cryptic biosynthetic gene clusters, and different strategies are there to trigger these gene pathways for the extensive study of natural product chemistry. Hence, the advanced technologies play a crucial role to achieve efficient discovery and productivity of novel microbial bioactive compounds. This chapter provides an outline on the mass production of microbial natural products derived from marine sponges and corals.
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The phylogenetic diversity of bacteria isolated on solid media from the Baltic Sea, Mediterranean Sea, Southern California Eight, Skagerrak, Weddell Sea (ice) and Andaman Sea was investigated by means of 16S rRNA gene sequence analysis. Of the 128 sequenced isolates, 52% showed similarity on the species level to previously reported bacteria, while as many as 18% showed a sequence similarity below 93%, which in the closest case would represent difference at the genus level. A majority of the isolated gamma-Proteobacteria could be assigned to known species, while half of the alpha-Proteobacteria were only identified to the genus level. Bacteria affiliated with the Flexibacter-Cytophaga-Bacteroides phylum showed the lowest levels of similarity to previously sequenced bacteria, mainly representing novel genera. Closely related isolates most often originated from the same geographic area. Nevertheless, our data also demonstrated that most genera have closely related representatives widely distributed between different sea areas. Isolates related to environmental clones, with a sequence similarity above the tentative genus level, were found in 51 cases, of which 17 were more similar to clones than to cultured bacteria. From this result we concluded that a large proportion of the great species richness of marine bacteria, found by culture-independent techniques, is likely to be verified through information from live and functional bacteria.
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Fluorescent Pseudomonas strains were isolated from 38 undisturbed pristine soil samples from 10 sites on four continents. A total of 248 isolates were confirmed as Pseudomonas sensu stricto by fluorescent pigment production and group-specific 16S ribosomal DNA (rDNA) primers. These isolates were analyzed by three molecular typing methods with different levels of resolution: 16S rDNA restriction analysis (ARDRA), 16S-23S rDNA intergenic spacer-restriction fragment length polymorphism (ITS-RFLP) analysis, and repetitive extragenic palindromic PCR genomic fingerprinting with a BOX primer set (BOX-PCR). All isolates showed very similar ARDRA patterns, as expected. Some ITS-RFLP types were also found at every geographic scale, although some ITS-RFLP types were unique to the site of origin, indicating weak endemicity at this level of resolution. Using a similarity value of 0.8 or more after cluster analysis of BOX-PCR fingerprinting patterns to define the same genotypes, we identified 85 unique fluorescent Pseudomonas genotypes in our collection. There were no overlapping genotypes between sites as well as continental regions, indicating strict site endemism. The genetic distance between isolates as determined by degree of dissimilarity in BOX-PCR patterns was meaningfully correlated to the geographic distance between the isolates' sites of origin. Also, a significant positive spatial autocorrelation of the distribution of the genotypes was observed among distances of <197 km, and significant negative autocorrelation was observed between regions. Hence, strong endemicity of fluorescent Pseudomonas genotypes was observed, suggesting that these heterotrophic soil bacteria are not globally mixed.
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Sponges (class Porifera) are evolutionarily ancient metazoans that populate the tropical oceans in great abundances but also occur in temperate regions and even in freshwater. Sponges contain large numbers of bacteria that are embedded within the animal matrix. The phylogeny of these bacteria and the evolutionary age of the interaction are virtually unknown. In order to provide insights into the species richness of the microbial community of sponges, we performed a comprehensive diversity survey based on 190 sponge-derived 16S ribosomal DNA (rDNA) sequences. The sponges Aplysina aerophoba and Theonella swinhoei were chosen for construction of the bacterial 16S rDNA library because they are taxonomically distantly related and they populate nonoverlapping geographic regions. In both sponges, a uniform microbial community was discovered whose phylogenetic signature is distinctly different from that of marine plankton or marine sediments. Altogether 14 monophyletic, sponge-specific sequence clusters were identified that belong to at least seven different bacterial divisions. By definition, the sequences of each cluster are more closely related to each other than to a sequence from nonsponge sources. These monophyletic clusters comprise 70% of all publicly available sponge-derived 16S rDNA sequences, reflecting the generality of the observed phenomenon. This shared microbial fraction represents the smallest common denominator of the sponges investigated in this study. Bacteria that are exclusively found in certain host species or that occur only transiently would have been missed. A picture emerges where sponges can be viewed as highly concentrated reservoirs of so far uncultured and elusive marine microorganisms.
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Barriers to dispersal between populations allow them to diverge through local adaptation or random genetic drift. High-resolution multilocus sequence analysis revealed that, on a global scale, populations of hyperthermophilic microorganisms are isolated from one another by geographic barriers and have diverged over the course of their recent evolutionary history. The identification of a biogeographic pattern in the archaeon Sulfolobus challenges the current model of microbial biodiversity in which unrestricted dispersal constrains the development of global species richness.
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Marine sponges (Porifera) harbor large amounts of commensal microbial communities within the sponge mesohyl. We employed 16S rRNA gene library construction using specific PCR primers to provide insights into the phylogenetic identity of an abundant sponge-associated bacterium that is morphologically characterized by the presence of a membrane-bound nucleoid. In this study, we report the presence of a previously unrecognized evolutionary lineage branching deeply in the domain Bacteria that is moderately related to the Planctomycetes, Verrucomicrobia, and Chlamydia lines of decent. Because members of this lineage showed <75% 16S rRNA gene sequence similarity to known bacterial phyla, we suggest the status of a new candidate phylum, named “Poribacteria”, to acknowledge the affiliation of the new bacterium with sponges. The affiliation of the morphologically conspicuous sponge bacterium with the novel phylogenetic lineage was confirmed by fluorescence in situ hybridization with newly designed probes targeting different sites of the poribacterial 16S rRNA. Consistent with electron microscopic observations of cell compartmentalization, the fluorescence signals appeared in a ring-shaped manner. PCR screening with “Poribacteria”-specific primers gave positive results for several other sponge species, while samples taken from the environment (seawater, sediments, and a filter-feeding tunicate) were PCR negative. In addition to a report for Planctomycetes, this is the second report of cell compartmentalization, a feature that was considered exclusive to the eukaryotic domain, in prokaryotes.
— We studied sequence variation in 16S rDNA in 204 individuals from 37 populations of the land snail Candidula unifasciata (Poiret 1801) across the core species range in France, Switzerland, and Germany. Phylogeographic, nested clade, and coalescence analyses were used to elucidate the species evolutionary history. The study revealed the presence of two major evolutionary lineages that evolved in separate refuges in southeast France as result of previous fragmentation during the Pleistocene. Applying a recent extension of the nested clade analysis (Templeton 2001), we inferred that range expansions along river valleys in independent corridors to the north led eventually to a secondary contact zone of the major clades around the Geneva Basin. There is evidence supporting the idea that the formation of the secondary contact zone and the colonization of Germany might be postglacial events. The phylogeographic history inferred for C. unifasciata differs from general biogeographic patterns of postglacial colonization previously identified for other taxa, and it might represent a common model for species with restricted dispersal.
A culture-independent molecular phylogenetic survey was carried out for the bacterial community in Obsidian Pool (OP), a Yellowstone National Park hot spring previously shown to contain remarkable archaeal diversity (S. M. Barns, R. E. Fundyga, M. W. Jeffries, and N. R. Page, Proc. Natl. Acad. Sci. USA 91:1609-1613, 1994). Small-subunit rRNA genes (rDNA) were amplified directly from OP sediment DNA by PCR with universally conserved or Bacteria-specific rDNA primers and cloned. Unique rDNA types among > 300 clones were identified by restriction fragment length polymorphism, and 122 representative rDNA sequences were determined. These were found to represent 54 distinct bacterial sequence types or clusters (> or = 98% identity) of sequences. A majority (70%) of the sequence types were affiliated with 14 previously recognized bacterial divisions (main phyla; kingdoms); 30% were unaffiliated with recognized bacterial divisions. The unaffiliated sequence types (represented by 38 sequences) nominally comprise 12 novel, division level lineages termed candidate divisions. Several OP sequences were nearly identical to those of cultivated chemolithotrophic thermophiles, including the hydrogen-oxidizing Calderobacterium and the sulfate reducers Thermodesulfovibrio and Thermodesulfobacterium, or belonged to monophyletic assemblages recognized for a particular type of metabolism, such as the hydrogen-oxidizing Aquificales and the sulfate-reducing delta-Proteobacteria. The occurrence of such organisms is consistent with the chemical composition of OP (high in reduced iron and sulfur) and suggests a lithotrophic base for primary productivity in this hot spring, through hydrogen oxidation and sulfate reduction. Unexpectedly, no archaeal sequences were encountered in OP clone libraries made with universal primers. Hybridization analysis of amplified OP DNA with domain-specific probes confirmed that the analyzed community rDNA from OP sediment was predominantly bacterial. These results expand substantially our knowledge of the extent of bacterial diversity and call into question the commonly held notion that Archaea dominate hydrothermal environments. Finally, the currently known extent of division level bacterial phylogenetic diversity is collated and summarized.
Sponge remains have been identified in the Early Vendian Doushantuo phosphate deposit in central Guizhou (South China), which has an age of ∼580 million years ago. Their skeletons consist of siliceous, monaxonal spicules. All are referred to as the Porifera, class Demospongiae. Preserved soft tissues include the epidermis, porocytes, amoebocytes, sclerocytes, and spongocoel. Among thousands of metazoan embryos is a parenchymella-type of sponge larvae having a shoe-shaped morphology and dense peripheral flagella. The presence of possible amphiblastula larva suggests that the calcareous sponges may have an extended history in the Late Precambrian. The fauna indicates that animals lived 40 to 50 million years before the Cambrian Explosion.
Comparative sequence analysis of small subunit rRNA is currently one of the most important methods for the elucidation of bacterial phylogeny as well as bacterial identification. Phylogenetic investigations targeting alternative phylogenetic markers such as large subunit rRNA, elongation factors, and ATPases have shown that 16S rRNA-based trees reflect the history of the corresponding organisms globally. However, in comparison with three to four billion years of evolution the phylogenetic information content of these markers is limited. Consequently, the limited resolution power of the marker molecules allows only a spot check of the evolutionary history of microorganisms. This is often indicated by locally different topologies of trees based on different markers, data sets or the application of different treeing approaches. Sequence peculiarities as well as methods and parameters for data analysis were studied with respect to their effects on the results of phylogenetic investigations. It is shown that only careful data analysis starting with a proper alignment, followed by the analysis of positional variability, rates and character of change, testing various data selections, applying alternative treeing methods and, finally, performing confidence tests, allows reasonable utilization of the limited phylogenetic information.