Characterisation of fungal-deficient mutant fm3 Growth curve




Дата канвертавання27.04.2016
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Characterisation of fungal-deficient mutant FM3

Growth curve


To ensure that the loss of antifungal activity was not due to a mutation in an essential pathway that would lead to decreased cell activity and growth, growth curves were performed in VNSS (41). The wild type P. tunicata and the transposon generated fungal-deficient mutant had essentially identical growth curves. Cells entered logarithmic growth after 5 hours and reached stationary phase after 15 to 20 hours. A graph of optical density versus time is shown in Figure S1.

Figure S1: Growth curve of P.tunicata wildtype and the transposon generated mutant FM3 recorded at an optical density of 610 nm over a period of 26 hours.

Biochemical testing


The wild type and the fungal-deficient mutant FM3 were both Gram negative rods which were oxidase and catalase positive and could not ferment carbohydrates (Table S1).
Table S1: A comparison of the wildtype P. tunicata and the transposon generated mutant.

Chemical Test

Wildtype

Transposon mutant

Gram Stain

Negative

Negative

Oxidase

Positive

Positive

Catalase

Positive

Positive

Hugh and Leifson

Negative

Negative

Antifouling activity of fungal-deficient strain FM3

Anti-bacterial activity and auto-inhibition of the fungal-deficient mutant


The ability of the fungal mutant to inhibit bacterial growth and display autoinhibitory activity was assessed using an agar overlay technique (35). The fungal-deficient mutant was found to display the same inhibition of the test strains as the wildtype P. tunicata (Table S2).
Table S2: Growth inhibition of bacteria in the presence of P. tunicata wild-type and mutant strain FM3.




Growth inhibition (mm)

Target bacterium

Wild-type

FM3

P. tunicata

3.5

3

Bacillus subtilis

3

3


Antilarval bioassays


Invertebrate larval settlement assays were performed as previously reported (31). In brief, bacterial biofilms were established on glass slides for 2 days, thereafter washed carefully twice with 1 ml of sterile seawater and added to a petri dish. Thirty ml of filtered (0.22 m) sterile seawater and around 20-30 larvae were added to each petri dish and incubated at 28C for 2 days. The number of larvae was counted under a dissecting microscope and the percentage settlement, as compared to the control settlement on non-treated surfaces, was determined. All assays were conducted in triplicate.
Table S2: Inhibition of invertebrate larval settlement due to the presence of P. tunicata wild-type and fungal-deficient mutant strain FM3. The control was a non-treated surface.


Hydroides elegans







% settlement

Standard Deviation

Control

69

7

P. tunicata

0

0

FM3

0

0




Balanus amphitrite







% settlement

Standard Deviation

Control

89

9

P. tunicata

0

0

FM3

0

0



Antialgal bioassays

Algal spore settlement assays were performed as previously reported (31). In brief, bacterial biofilms were established in 24-multi-well culture plate (Sigma) with each well containing 1 ml of the VNSS medium for assessing the effects against spores of the green alga U. australis or on 36-mm Petri dishes containing 3 ml of VNSS medium for determining inhibition of settlement of spores of the red alga Polysiphonia sp. Biofilms were developed at 28C for 2 days and washed before fresh sterile seawater was added to each well prior to the algal bioassay. All assays were conducted in triplicate.



Table S3: Inhibition of algal spore settlement due to the presence of P. tunicata wild-type and fungal-deficient mutant strain FM3. The control experiments were treated with seawater alone.


Polysiphonia sp.







% settlement

Standard Deviation

Control

83

10

P. tunicata

2

2

FM3

3

5




Ulva australis







% settlement

Standard Deviation

Control

97

4

P. tunicata

0

0

FM3

0

0


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