bacterial co-culture: exploring microbial interactions for

Fleurdeliz Maglangitab*, Rainer Ebela, Hai Denga

*ffmaglangit@up.edu.phaMarine Biodiscovery Centre, University of Aberdeen, Aberdeen, Scotland, UK bUniversity of the Philippines, Lahug, Cebu City, Philippines

Conclusion

References Acknowledgements1 FEMS Microbiol. Rev., 2014, 38, 90–1182 FEMS Microbiol. Lett., 2010, 309, 1–73 Chem. Biol., 2014, 21, 1211–1223

Methodology

Background

Interactions among microorganisms are fundamental to microbial ecosystem dynamics 1. Bacteria live in communities and theydepend on other organisms to grow and reproduce. This can be best illustrated by the so-called - “great plate count anomaly” 2, whichclaims that only 1% of the known organisms can be readily cultivated on its own using standard laboratory methods. In this study, wecultured two bacterial species, Streptomyces sp. MA37 and Pseudomonas sp. that are physically separated by a semi-permeablemembrane yet allows exchange of nutrients, and dissolved or colloidal chemical signals – thus, a chemical communication between twointeracting species.

AIMS:• To determine whether the co-culture cultivation will produce new specialized metabolites compared to bacterial monocultures• To isolate and identify the structure of these new metabolites

Results

Results

Figure 4. Structure of compound 1 with COSY (▬) and key

HMBC (→ ) correlations

Position 1H mult. (J, Hz) 13C

1 10.50 (OH)

2 6.98 (8.2) 111.4

3 7.13 (7.8) 121.3

4 8.40 (7.8) 115.5

4a - 123.0

4b - 115.9

4c - 120.3

5 - 171.7

6

7 - 171.7

7a - 120.3

7b - 115.9

7c - 123.0

8 8.40 (8.2) 115.5

9 7.13 (7.8) 121.3

10 6.98 (7.8) 111.4

11

11a - 130.7

12 11.59 (NH)

12a - 129.7

12b - 129.7

13 11.59 (NH)

13a - 130.6

Streptomyces sp. MA37

Pseudomonas sp.

0.22 µm membrane

Co-culture Fermentation

The organism is cultured in each chamber filled with up to

300mL of ISP2 media separated by a 0.22µm membrane filter.

No direct contact but ensuring the exchange of metabolites.

Solid Phase Extraction (SPE) HPLC Purification LCMS/NMR Structure Elucidation

Future Work

Figure 2. HPLC Profile (3D overlay) of A. Co-culture B. Streptomyces sp. MA37, and C. Pseudomonas sp.

monocultures (* new peaks found in co-culture that are not in A or B)

A. co-culture

B. Streptomyces MA37 monoculture

C. Pseudomonas monoculture

*

*

* ***

*

**

*

* ** * *

UV chromatogram of Compound 1

Figure 1. Natural Product Discovery Workflow

Figure 3. Bioassay results of co-culture extract and SPE fractions, and monocultures of Streptomyces sp. MA37 and

Pseudomonas sp.

Bacterial Co-culture: Exploring Microbial Interactions for the Production of Specialised Metabolites

Many metabolites were absent in the monocultures wereco-culture specific (Fig. 2), hence microbial interactions elicitthe production of these natural products 3.

Compound 1 is an indolocarbazole alkaloid (Fig. 4, Table 1). Onthe basis of LC MS analysis, it was confirmed that thiscompound is co-culture specific. Neither bacteria produced thismetabolite when cultivated alone.

Table 1. 1H and 13C-NMR of Compound 1 in

d-DMSO at 600MHz

Using bioassay-guided screening (Fig. 3), we were able totarget metabolites for isolation.

The active extract was fractionatedusing SPE followed by semi-prepreversed-phase C18 HPLC, and thestructure was elucidated byspectroscopic techniques including1D, 2D NMR and LC MS.

Co-culture is an efficient method to elicit production of novelantimicrobials through species interactions. This approach paves theway for increasing the chemical diversity of microbes when grown invitro.

• Elucidate and characterize the structures of isolated compounds• Test the bioactivity of pure compounds

FRASDP for the PhD student fellowshipLeverhulme Trust-Royal Society Africa award (AA090088)

- no data available