1. Bacteria From Pseudo-nitzschia multiseries and Mechanisms By Which They May Influence Toxin Production
Gallacher, S., Macrae, J., Ferguson, C., Smith, E.A., Hess, P., Johnstone, M., Glover, L.A., Bates, S.S., & Garthwaite, I.

2. Axenic Pseudo-nitzschia multiseries cultures produce DA but at lower concentrations than cultures with bacteria
Addition of bacteria to axenic Pseudo-nitzschia cultures produces fold increase in DA concentrations
Enhancement varies depending on bacterial strain and the diatom culture
Mechanism is unknown

3. Potential mechanisms Autonomous production Indirect effects
Regeneration
of nutrients
Precursors
Indirect
signals

4. Pseudo-nitzschia culture
4 dominant bacteria
yellow, pink, orange, cream
Roseobacters and Caulobacters
Lag phase
Stationary phase

5. Bacterial growth

6. Do these strains produce DA?
ELISA: DA antibody determination limit in these assays = ng/ml
Problems with the solvent in the standard curves
DA signals not detected if samples were diluted to any extent

7. ELISA results for yellow strain

8. Analysis by HPLC (Quilliam et al
Analysis by HPLC (Quilliam et al., 1995 determination limit = 10 ng/ml)
HPLC analysis of bulk culture: DA not detected!
Tryptophan interference noted
Was tryptophan causing false positives in the ELISA?
Could we use media without tryptophan?
Quantity of DA too low?
Losses during the clean-up stage?

9. Tryptophan Was present in the medium used
Did not interfere with the ELISA
Did interfere with DA peak by HPLC
Strains did not grow in f/2

10. Recovery of DA from bacterial cells

11. Recovery of DA from bacterial supernate

12. Negative DA result may have been due to difficulties with HPLC methodology
Is it possible to increase DA concentration as determined by ELISA?

13. DA and bacteria

14. Bacteria/plant cell-cell communication
Bacteria respond to external signals from neighbouring cells
Signalling
molecules
cell surface
components
Secreted proteins s
Bacteria function in a multicellular manner
Maintain plant-bacterial interaction
Can manipulate eukaryotic host cell transduction pathways

15. N-acyl-homoserine lactones
Chromobacterium mutant = no pigment
+ HSL
Purple pigment (4-8C or 10-14C)
E. coli (lux gene) + HSL = Bioluminescence (6-10C)

16. HSL’s and plasmids
Mutation of strains will provide more information

17. Conclusions ELISA infers DA may be present in bacteria
HPLC methodology for DA bacterial analysis requires modification
Adhesion increases “DA” by ELISA
HSL’s may be present in some strains
Most of the strains carry plasmids
More research required to determine if HSL and plasmids are important