This study aimed to induce lysogenic phages in Oenococcus oeni bacteria and determine their host range. Various induction methods revealed that 11 of 25 bacterial strains carried phages. Mitomycin C was the most effective at inducing phages. Two strains showed no growth inhibition after induction but still released phages, indicating a lack of correlation between inhibition and phage release. The phages induced were similar in morphology to other reported O. oeni phages. This research provided new insights into phage induction methods and identified strains suitable or risky for malolactic fermentation. Future work could help clarify relationships between induction mechanisms and inhibition/phage release.

1. C-1
Part 3 (Chapter 4)
The Occurrence of Bacteriophages in
Oenococcus oeni

2. C-2
Aims of this study
1. To induce lysogenic phage by different
induction methods
2. To determine phage-host range

3. C-3
Story-- how did I find it?
Aug 2006
Oe vs Oe – Looking for potential
producer of bacteriocins

4. C-4
THKOe03 THKOe05
Indicator: THKOe20

5. C-5
Phage
carrier:THKOe24
Indicator: THKOe01
Later, I proofed that THKOe24 released phages (Φ24)
after heat induction, not released phage spontaneously.

6. C-6
Materials and Methods
Cultures: 25 strains of O. oeni
Medium: MLB with 1 mmol l-1
CaCl2

7. C-7
Induction methods
1 Mitomycin C (MC)
2 Thermal induction
3 UV irradiation

8. C-8
Induction methods
1 Mitomycin C (MC)
2 Thermal induction
3 UV irradiation
(Structure source: Wikipedia,
public domain)
Stock solution: 1 mg ml-1
Final concentration: 1 μg ml-1

9. C-9
Induction methods
1 Mitomycin C (MC)
2 Thermal induction
3 UV irradiation
Water bath: 78C°C for 1 min (temperature inside
reached 50°C)(why this condition?)
--> Imitating the conditions of molten agar for overlay

10. C-10
Induction methods
1 Mitomycin C (MC)
2 Thermal induction
3 UV irradiation
20 cm
For 2
min
Phillips TUV64 T5, 75W UV-C

11. C-11
48 hr after induction, spotting

12. C-12
Flow chart
Induction
A: MC
B: Thermal
C: UV
D: Control
Growth
OD600,
48 hr
Preparing
indicator
strains
Centrifuge
Spotting

13. C-13
A. 11 strains out of 25 are phage carriers
3 strains-- released phage spontaneously
8 strains-- phages released after induction
11/25 = 44% are carrying phages
Results

14. C-14
B. High sensitivity strains
THKOe01, 11, and 22 were phage-free
strains, but sensitive to eight or nine phages.
--> Bad for MLF (high risk strains)

15. C-15
C. Low sensitivity strains
THKOe02, 08, 09 and 16 were not sensitive
to any of 11 phages in this test, and they
were not phage carriers
--> Suitable for MLF (low risk, safer)

16. C-16
D. Phage carrier but sensitive to other
phages
1. Φ13 caused plaques on phage carrier,
THKOe03, 04, 05 and 06
2. Some phage-carrying strains were not sensitive
to other phages: THKOe12, 13, 14, 15.
3. But all phage-carrying strains were resistant to
the phage released by themselves.

17. C-17
Results
Phage and lytic host range. Indicators strains may form lytic plaques from lysates with various treatments.
Phage and
induction
Indicator strains*
01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Φ03 MC ++ - - - ++ - - - - - ++ - - - - - - - - - - ++ - - -
T ++ - - - ++ - - - - - ++ - - - - - - - - - - ++ - - -
UV ++ - - - ++ - - - - - ++ - - - - - - - - - - ++ - - -
C ++ - - - ++ - - - - - ++ - - - - - - - - - - ++ - - -
Φ04 MC - - - - - - - - - - - - - - - - - - - - - - - - -
T ++ - - - - - - - - - - - - - - - - - - - - - - - -
UV ++ - - - - - - - - ++ - - - - - - - - - - - - - - -
C - - - - - - - - - - - - - - - - - - - - - - - - -
Φ05 MC ++ - - - - - + - - - ++ - - - - - ++ ++ - ++ ++ ++ - - -
T - - - - - - - - - - + - - - - - - + - + + - - - -
UV + - - - - - - - - - + - - - - - + + - + + + - - -
C + - - - - - - - - - + - - - - - + + - + - - - - -
Φ06 MC ++ - - - - - - - - - - - - - - - - - - - - - - - -
T - - - - - - - - - - - - - - - - - - - - - - - - -
UV - - - - - - - - - - - - - - - - - - - - - - - - -
C - - - - - - - - - - - - - - - - - - - - - - - - -
Φ12 MC + - - - - - - - - - + - - - - - + + - + + + - - -
T - - - - - - - - - - - - - - - - - - - - - - - - -
UV - - - - - - - - - - - - - - - - - - - - - - - - -
C - - - - - - - - - - - - - - - - - - - - - - - - -
CH4, Table 4.1

18. C-18
(Continued)
Phage and
induction
Indicator strains
01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Φ13 MC ++ - ++ ++ - ++ - - - - - - - - - - - - - - - ++ ++ - -
T ++ - - - - - - - - - - - - - - - - - - - - - - - -
UV ++ - - - - - - - - - - - - - - - - - - - - ++ - - -
C ++ - - - - - - - - - - - - - - - - - - - - - - - -
Φ14 MC + - - - - - - - - - + - - - - - + + - + - + - - -
T - - - - - - - - - - - - - - - - - - - - - - - - -
UV - - - - - - - - - - - - - - - - - - - - - - - - -
C - - - - - - - - - - - - - - - - - - - - - - - - -
Φ15 MC + - - - - - - - - - + - - - - - + + - + - + - - -
T - - - - - - - - - - - - - - - - - - - - - - - - -
UV - - - - - - - - - - - - - - - - - - - - - - - - -
C - - - - - - - - - - - - - - - - - - - - - - - - -
Φ19 MC - - - - - - - - - - + - - - - - + + - + - + - - -
T - - - - - - - - - - - - - - - - - - - - - - - - -
UV - - - - - - - - - - - - - - - - - - - - - - - - -
C - - - - - - - - - - - - - - - - - - - - - - - - -
Φ24 MC ++ - - - - - - - - + - - - - - - - - - - + - - -
T ++ - - - - - - - - - - - - - - - - - - - - - - - -
UV ++ - - - - - - - - - - - - - - - - - - - - - - - -
C - - - - - - - - - - - - - - - - - - - - - - - - -
Φ25 MC - - - - - - - - - - + - - - - - + + - + - - - - -
T - - - - - - - - - - - - - - - - - - - - - - - - -
UV - - - - - - - - - - - - - - - - - - - - - - - - -
C - - - - - - - - - - - - - - - - - - - - - - - - -

19. C-19
E. The first reported MC non-inducible phage
THKOe04
MC
THKOe03
T
UV
C
MC
T
UV
C
Indicator: THKOe01
THKOeΦ04

20. C-20
THKOe04
MC
THKOe03
T
UV
C
MC
T
UV
C
Indicator THKOe10 was only sensitive to phage
induce by UV irradiation
Indicator: THKOe10

21. C-21
THKOe06
MC
THKOe05
T
UV
C
MC
T
UV
C
Mitomycin C was more effective
for induction
Few: Control
Few: Heat
More: UV
A lot of: MC

22. C-22
Phage morphology
1. Phage propagation
3-time amplification
2. Electron microscope
(supported by Dr. Hoch)
3. Measuring dimensions
(software: ImageJ)

23. C-23
Diameter
Taillength
36-55 nm
129-211 nm

24. C-24
So far, all phages of O. oeni are
reported as type B phage, including
phages isolated in this study.

25. C-25
Bradley 1967

26. C-26
Correlation:
Releasing phage
VS
Growth inhibition

27. C-27
Poblet-Icart et al. 1998
Review
Poblet-Icart et al. (1998)
Three types of results
after MC induction
They consider that the
growth inhibition
represents “lysogeny”
They reported that 17
phages were identified
out of 19 strains with
“inhibition”

28. C-28
In this study, according MC inhibition
Group A: No inhibition by MC
Group B: Moderate inhibition
Group C: Significant inhibition
*Remember, we did not discuss UV and thermal
induction yet.

29. C-29
Group A
no inhibition
no phage found
Strain: 01, 07,
18, and 22
Group B
inhibition
no phage
found
Strain: 02, 04,
08, 09, 10, 11,
17, 20, and 23
Exception: Φ04
Group C
big inhibition
phage found
Strain: 03, 05,
06, 12, 13, 14,
15, 16, 19, 21,
24, and 25
Exception: 16
and 21
Big inhibition
no phage found
strain: 21
CH4, Figure 4.1

30. C-30
About growth inhibition and phage releasing
1. Poblet-Icart et al. (1998) think that is highly correlated.
--> 17 of 19: 2 due to no appropriated host.
2. In this study, results are similar to their's
3. But, in this study, UV and heat never inhibited the
growth while some phages were induced (THKOe04, 05, 13,
and 24 )
--> Different mechanism? Coincidence?

31. C-31
My opinion
1. For UV irradiation and thermal induction:
No correlation
2. For MC induction: correlation is not high

32. C-32
Conclusion
1. The first MC non-inducible phage
reported.
2. The results offer more induction methods
that are good for culture companies to
investigate lysogeny and prepare phage-
free cultures.

33. C-33
Conclusion
3. The results show high and low risk
strains and that are useful for culture
companies to prepare “suitable” cultures
but there is a saying.....
4. Morphology: the same as other reports

34. C-34
Future work
1. More induction methods
2. Induction mechanisms
3. To clarify growth inhibition and phage-
release
4. Test in real wines or synthetic wines.

35. C-35
Acknowledgment
Dr. Henick-Kling, Dr. Padilla-Zakour, Dr. Worobo
Lallemand Inc.New York Wine and Grape Foundation,
The Ministry of Education, Taiwan
John Churey, Kathy Arnink, Luann Preston-Wilsey,
Debby Ditzell, Penny Lynn, Ed Lavin, Dr. Siebert, Dr.
Acree, Dr. Hoch and all people who have helped me.