Expression of biosynthetic gene cluster in sponge associated bacterium during co-cultivation experiment: Chemical warfare between bacterial neighbors

pcaQ pcaD/C pcaH pcaG pcaI pcaJ pcaF pcaB peptidase

0 1 2 3 4 5 6 7 8 9kbkb10

Narsinh L. ThakurNational Institute of Oceanography, Goa

Vladislav A. Grebenjuk and Werner E. G. Müller, UNI - Mainz, Germany

Agenda

Sponge-associated bacteria

Biosynthetic gene cluster

PKS gene pathway

Co-cultivation experiment

PCA gene pathway

Summary

Sponge – bacteria association

Sponge-bacteria association: a fascinating research topic

In some sponge species 40% of the animal‘s biomass is attributed tobacteria

Elicited considerable interest as sponges are rich in bioactivemetabolites

Thakur and Müller Symbiosis, 2005, 39: 1- 8

Sponge

Classification:Phylum: PoriferaClass: DemospongiaeOrder: HadromeridaFamily: SuberitidaeGenus: SuberitesSpecies: domuncula

Sponge Suberites domuncula

Species: domuncula

Collected from Rovinj, Croatia

Isolation of bacteria

Suberites domuncula

Bacterial colonies

SB1RFLP pattern

alphaalpha--proteobacteriumproteobacterium MBICMBIC 33683368

Antibacterial and anticancer activity

SB1SB2SB3SB4SB5SB6

Thakur et al. Aquatic Microbial Ecology, 2003, 31: 77-83

SB2 bacterium

SB2 strain (α- Proteobacterium MBIC 3368) has beenisolated from different sponges irrespective of theirtaxonomic identity or geographical location.

Bacterial strain SB2 showed antibacterial and cytotoxicpotential.

The genome of bacterial strain SB2 contains gene clusterfor polyketide synthases.

Biosynthetic gene pathways

Natural genetic machinery involved in the production ofcompounds and energy in the living organisms.

Polyketides and Non-ribosomal peptides are the mostimportant biosynthetic pathways.important biosynthetic pathways.

Polyketides and Nonribosomal Peptides arepharmacologically important as antibacterial, cholesterolcontrol, anticancer, immunosuppressant drugs as well aspigments, toxins, nitrogen storage polymers etc.

Biosynthetic gene pathwaysPKS I (Modular Polyketide Synthases)

Module I Module II Module III Module IV

Extender Moduleloading module

ketosynthase (KS) domain

Acyl transferase (AT) domain

acyl carrier protein (ACP)

thioester domain

Detection of PKS gene pathway

DNA

isolation

Primer design and

PCR

SB2

Detection of KS-3 gene on gel

KS-1KRKS AT DH ER

14 16 18 20 22 24 26 28 30 32kbkb

34 36 38

KS-2 KS-3 KS-4TE

Cloning

Restriction

digestion

DNA

isolationDNA fragement

Vector

Restriction

digestionCosmidVector

SB2

Müller et al., Applied & Environmental Microbiology, 2004, 70: 2332-2341

Recombinant Plasmid

Transformation in bacterial hostclones

E. coli

Selected clones with PKS cluster

Chemical warfare

SB2 SB6alphaalpha--proteobacteriumproteobacterium gamagama--proteobacteriumproteobacterium

Growth inhibition

Co-cultivation experiment

Filter paper 0.22 µm

SB6

MediumSB2

SB6

Co-cultivation experiment

After 72 h incubation

SB2 bacterium

Real-time PCR analysis

After 24 h, 48 h, 72 h incubation

RNA

extraction cDNA synthesis

Real-time Q-PCR analysis

Detection of KS-3 gene expression

Gene expression

KS-3 gene

150

200

ssio

n (%

) 5 - fold dilution of cDNA

25- fold dilution of cDNA

Up-regulation after 24 h incubation

SB2 SB2 + SB6 SB2 + SB10

50

100

Rel

ativ

e ex

pres

Gene expression and growth curve

SB2

1

2

3

4

5

OD

600

Highest up-regulation during late exponential phase and early stationary phase, which is reported to be a peak period of secondary metabolite production in bacteria.

0

1

3h 6h 9h 12h 15h 18h 21h 24h 27h 30h 33h 36h 48h 54h 72h

time interval

PCA Gene cluster

SB2 showed very slow growth in normal Zobell medium

While exploring pks gene cluster Protocatechuate gene cluster

(pca) was detected

PCA gene cluster was cloned and sequenced

PCA gene cluster comprises all the genes coding for enzymesg p g g y

involved in the conversion of protocatechuate to acetyl coenzyme A.

pcaQ pcaD/C pcaH pcaG pcaI pcaJ pcaF pcaB peptidase

Protocatechuate pathway

0 1 2 3 4 5 6 7 8 9kbkb10

Growth study

Growth of SB2 in M9 medium supplemented with

Glucose

Sodium benzoate

Protocatechuate 0.25

SOBPCAGlucose

3 6 9 12 24 27 30 33 36 48

0.05

0.10

0.15

0.20

OD

600

Time (hrs)SB2

Müller et al., Applied & Environmental Microbiology, 2004, 70: 2332-2341

(Concentration 4 mM)

Summary

Association of alpha-proteobacterium (MBIC 3368) withsponges

Role of biosynthetic gene cluster in the chemical warfarebetween bacterial neighborsbetween bacterial neighbors

Gene up-regulation during late exponential phase andearly stationary phase, which is reported to be a peakperiod of secondary metabolite production

Thank you