Diapositivo 1 - Weebly

shamebagBiotechnology

Feb 22, 2013 (4 years and 3 months ago)

273 views

Please note:

This is a scientific communication for personal
use only. All data is an intellectual property of
Ana I. S. Esteves. Please do not copy or use
without consent. For more info or to obtain
permission, please email me at
aidsesteves@gmail.com


Thank you

Marine sponges

and their symbionts:


soulmates or partners by chance?

Ana I. S. Esteves

Microbial Ecology and Evolution

Centre of Marine Sciences of Algarve, Portugal

IAAC Jena, 28th September 2011

Marine Sponges

Ancient multicellular animals

No cell differentiation

Totipotency

Benthic filter feeders

High gene density

Diverse morphologies and habitats

Essentially chemically defended

Extremely sociable!

IAAC Jena, 28th
September

2011

Marine Sponge Biotechnology

Blunt

et
. al. 2009
Nat
.
Prod
.
Rep
. 26, 170
-
244

Sponges are the most prolific producers of new compounds


Diverse applications:

-

Pharmaceuticals

Antivirals, antitumorals, antibiotics, analgesics, anti
-
inflamatories…


-

Technology

Anti
-
fouling, biomaterials, bioremediation…


Scale
-
up problem

Host or symbiont?

IAAC Jena, 28th September 2011

Taylor et. Al 2007 Microbiol. Mol. Biol. Rev. 71(2): 295
-
347

IAAC Jena, 28th September 2011

Sponge hidden microbial world

Microbes can make up to 40% of the sponge volume


Less than 1% has been cultivated


Dense, diverse and specific microbial communities


Beneficial to host


carbon fixation, chemical defense…


IAAC Jena, 28th September 2011

„Unlocking the bacterial diversity

encrypted in the metagenomes of marine sponges“

Determine bacterial diversity in marine sponges of the family
Irciniidae


Reveal the interplay between the host and its biogeographical

setting

in shaping the
structure of these communities


Unveil antimicrobial properties of culturable bacteria from
Irciniidae

specimens

Objectives:

Ircinia variabilis

Sarcotragus spinosulus

Algarve

ALG

MAD

AZO

IAAC Jena, 28th September 2011

„Unlocking the bacterial diversity encrypted in the metagenomes of marine sponges“

Methodology

Culture
independent
approach

Culture
dependent
approach

IAAC Jena, 28th September 2011

454 Pyrosequencing

-
DNA

is

cut

in

fragments

(or

specific

PCR)

and

single

stranded


-
Each

fragment

adheres

to

one

bead


-
Fragment

is

amplified


1

bead

=

millions

of

copies


-
Each

bead

goes

into

one

well


-
Sequencing

takes

place


Advantages over DGGE:

-
You get sequences and not bands

-
You can know what each sequence is

-
High Throughput: no need for cut
-
and
-
clone bands

-
400k sequences per run


(in our case: 6000


9000 seqs/sample)

IAAC Jena, 28th September 2011

A glimpse on pyrosequecing results…

„Unlocking the bacterial diversity encrypted in the metagenomes of marine sponges“

sponge

homogenate

Plate washing

4 dominant phyla

Acidobacteria and
Actinobacteria enriched sponges

Cyanobacteria and Flavobacteria
“de
-
selected” in sponge vs
seawater

IAAC Jena, 28th September 2011

„Unlocking the bacterial diversity encrypted in the metagenomes of marine sponges“

Irciniidae

in a bottle: culturable bacteria from
Ircinia sp.

and
Sarcotragus sp.


Marine
Broth

+

2.5 g

25 mL


CMFASW

25 CFU/sponge sample

Marine Agar

Centrifuge

10.000 g

5 min

̴
3
days

DNA
extraction

Homogenate

10
Sarcotragus sp.


4
Ircinia sp.

16S rDNA PCR

327 isolates


155 ≠ genotypes

BOX
-
PCR
profiling

IAAC Jena, 28th September 2011

16S rDNA Diversity

DNA of isolated
bacteria

PCR amplification with primers
specific for (bacterial) 16S

Check in agarose gel

Sequence electropherogram

RDP Classifier

ARB

NCBI BLAST

IAAC Jena, 28th September 2011

BOX
-
PCR Genotyping

Same 16S rDNA sequence
DOES NOT MEAN

same strain!


BOX element
-
154 bp highly conserved repetitive DNA sequence,
located in distinct,


intergenic positions around the genome of bacteria


PCR with specific (single) primer

Bac. A

Bac. B

F3

F2

F1

F5

F4

Agarose gel

IAAC Jena, 28th September 2011

BOX
-
PCR Genotyping

Microbulbifer sp.
BOX
-
PCR Fingerprint

17 isolates

4 different genotypes

97.7
75.8
99.1
99.0
98.1
97.8
99.0
95.8
70.6
95.6
60.3
98.7
99.5
98.7
97.8
99.7
95.7
72.1
20.0
93.1
1.0
Microbulbifer
100
80
60
40
20
BOX-PCR
Key
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Isolate nº
244
245
250
34
41
39
45
45
45
45
317
327
105
136
346
350
353
42
44
320
Standard
Standard
Source
Alg10/17
Alg10/17
Alg10/17
Alg10/8
Alg10/8
Alg10/8
Alg10/8
Alg10/8
Alg10/8
Alg10/8
Alg10/20
Alg10/20
Alg10/11
Alg10/12
Alg10/21
Alg10/21
Alg10/21
Alg10/8
Alg10/8
Alg10/20
Gene Ruler
Gene Ruler
Gelcompar

IAAC Jena, 28th September 2011

Culturable Bacteria Diversity

Firmicutes
Actinobacteria
Bacteroidetes
Proteobacteria
Distribution per Bacterial Phyla

Alpha

Gamma

„Unlocking the bacterial diversity encrypted in the metagenomes of marine sponges“

Irciniidae

in a bottle: culturable bacteria from
Ircinia sp.

and
Sarcotragus sp.


-
Dominance of
Proteobacteria


-
Reproducible both for
Ircinia sp.
and
Sarcotragus sp.


-
Pseudovibrio

sp
.
,

Vibrio

sp
.

and

Ruegeria

sp
.

most

abundant

and

transversal

to

both

sponge

species



core

culturable

community?


-
Considerable

higher

diversity

in

Sarcotragus

sp
.


Distribution per Bacterial Genera

Ircinia sp.
Sarcotragus sp.
16S rDNA phylogeny for
Aquimarina sp.
(ARB software; forward sequences only)

IAAC Jena, 28th September 2011

„Unlocking the bacterial diversity encrypted in the metagenomes of marine sponges“

Irciniidae

in a bottle: culturable bacteria from
Ircinia sp.

and
Sarcotragus sp.


12 potential new species

Related to
Amphritea, Aquimarina,
Endozoicomonas, Pseudovibrio
and
Pseudoalteromonas


1 potential new genus

Related to
Endozoicomonas

Ircinia
variabilis

Sarcotragus
spinosulus

Sarcotragus
spinosulus

16S rDNA phylogeny for
Endozoicomonas sp.
(ARB software; forward sequences only)

IAAC Jena, 28th September 2011

„Unlocking the bacterial diversity encrypted in the metagenomes of marine sponges“

Irciniidae

in a bottle: culturable bacteria from
Ircinia sp.

and
Sarcotragus sp.




Biotech Potential

Polyketide Synthase (PKS) & Non
-
ribosomal Syntethase (NRPS) Genes

Pseudovibrio:

98% PKS+

48% NRPS+

20% NRPS++

Ruegeria:

25% PKS+

11
% NRPS+

Vibrio:

31% PKS+

3% NRPS+

Aquimarina:

92% PKS+

51% homology with type I PKS
from marine
Mycobacterium sp.

IAAC Jena,
28
th September
2011

„Unlocking the bacterial diversity encrypted in the metagenomes of marine sponges“

Irciniidae

in a bottle: culturable bacteria from
Ircinia sp.

and
Sarcotragus sp.


Whole
-
cell Antagonistic Assay

Competition of bacterial strains by the use of bioactive metabolites

Low activity


No reproducibility

Rethink assay!

IAAC Jena,
28
th September
2011

Taking one step further…

Functionality

Deciphering the codes of communication
between marine sponges and their symbionts:


an integrative metabolomics
-
transcriptomics approach

IAAC Jena, 28th September 2011


Deciphering the codes of communication between marine sponges and their symbionts


IAAC Jena, 28th September 2011


Deciphering the codes of communication between marine sponges and their symbionts


IAAC Jena,
28
th September
2011


Deciphering the codes of communication between marine sponges and their symbionts


Fresh from the lab: Biofilm formation

IAAC Jena, 28th September 2011


Deciphering the codes of communication between marine sponges and their symbionts


What is a biofilm?

Matrix

of

Extracellular

Polymeric

Substances

(EPS)



polysaccharides,

protein

and

DNA



that

surrounds

and

serves

as

support

and

protection

for

bacteria,

promotes

cell

adhesion

and

difusion

of

chemical

signals

.

IAAC Jena, 28th September 2011


Deciphering the codes of communication between marine sponges and their symbionts


“Bacteria

growing

in

a

surface
-
attached

biofilm

have

increased

production

of

chemical

defense

mechanisms

as

compared

to

planktonic

growth
.

This

may

represent

an

adaptive

response

to

compensate

for

the

loss

of

the

ability

to

escape

predation

when

competing

under

stressful

conditions

such

as

those

found

inside

the

sponges
.



Santos, O.C.S., et al. (2010), Research in Microbiology,
161
(7): 604
-
612

Why study biofilm formation?

“Bacterial

biofilm

formation

can

be

related

to

quorum
-
sensing,

antibiotic

resistance

and

adhesive

properties
.



StepanoviĆ, S., et al. (2007), APMIS
115
(8): 891
-
899

IAAC Jena, 28th September 2011


Deciphering the codes of communication between marine sponges and their symbionts


Biofilm

producing

bacteria

adhere

to

wells,

planktonic

bacteria

stay

in

suspension

Adherent

cells

stick

to

wells,

planktonic

bacteria

are

washed

away

Adherent

bacteria

nuclei

are

stained
;

non
-
binding

dye

will

be

washed

away

Concentration

of

dye

is

proportional

to

number

of

cells,

which

in

turn

is

proportional

to

quantity

of

biofilm

IAAC Jena, 28th September 2011


Deciphering the codes of communication between marine sponges and their symbionts


Results
:


-
69
%

of

isolates

produce

biofilm


-
8

strong

biofilm

producers

(without

sponge

extract)
:

Microccocus

sp
.
,

Pseudovibrio

sp
.

and

Ruegeria

sp
.


-
43
%

biofilm

enhancement

with

sponge

extract
;

strongly

enhanced
:

Microccocus

sp
.

(Mc
110
)

and

Ruegeria

sp
.

(Rg
351
)


-
8
%

slight

biofilm

inhibition

with

sponge

extract
:

Aquimarina

sp
.
,

Pseudovibrio

sp
.

and

Vibrio

sp
.


-
No

correlation

between

biofilm

formation

and

cell

growth

(OD

620

nm)

180 uL Bacteria
culture

20
uL
Sponge

Extract

Protocol adaptation:

200 uL Bacteria
culture

Protocol:

IAAC Jena, 28th September 2011


Deciphering the codes of communication between marine sponges and their symbionts


To be continued…

-
Minimum sponge extract concentration needed to enhance biofilm


-
Response of biofilm formation to synthetic AHLs


-
Sponge extract biofilm assay guided fractionation


-
Changes in the exo
-
metabolome of bacteria in response to sponge
extract


-
AHL screening in sponge isolated bacteria


-
Preliminary chemotaxis screening

IAAC Jena,
28
th September
2011

The Microbial Ecology and Evolution Research Group!

Rodrigo Costa,
the Boss!

Cristiane Cassiolato,
PhD student

?

Port wine!!!

Yeah!!!


IAAC Jena, 28th September 2011

Acknowledgements


€€€

Portuguese Science and Technology Foundation (FCT)
€€€€

Project PTDC/MAR/
101431
/
2008


CCMAR/FCT: Post
-
doctoral Fellowship CCMAR/BPD/
0002
/
2011


Our divers/sponge taxonomists: Jorge Gonçalves & Group (CCMAR)


Joana Xavier and Francisco Pires (University of Azores)


IAAC


University of Jena

Thomas Wichard

Dankeschön!