p1 (‑1mg) pKKT427

M 1

Fig. 5. P‑BKatE

(0.8%

agaroSe, 100V,

30min).

^{Lane M: i} EcoTmarker, ^lane 1:P‑BKatE.

3.3 Construct destination Vector

Constructed the destination vector PGUOO 1.

3.3.1 Digest pKKT427

Digested pKKT427 with StuI

(Takara)

at 37oC for 16 hours.

Plasmid pKKT427 is showed in Fig. 6.

The components of reaction:

2 p1 10xMBuffer l3 BllddH20

I.5 Lil StuI

Lined pKKT427 ^was purified with PEG.

Fig. 6. The map ofplasmid pKKT427.

3.3.2 Construct destination _vector pGUOO1

1. Ligation lined pKKT427 with rfcA

(Gateway

reading &ame

cassetteA)

Ligated lined _pKKT427 ^with rfcA

(‑1.7kb)

at 16oC for 30min with DNA Ligation Kit. The components of reaction:

1 Lil lined pKKT427

3 pl Gateway reading frame cassetteA

4 pl ligate solution ^.

2. Transformation One Shot ccdB Survival TIR E. coli.

Add 2 pl ligated mixture into One Shot ccdB Survival TIR E. coli.

spread the 50 pl culture selective plates

(cmR)

and incubate

ovemight

^{at 37oC.}

3. Check the plasmid pGUOO1 with ^PCR.

Po1ymerase: GO Taq

Template: PGUOO I

Cycling step:

Pre‑denaturation ^:

Denaturation ^:

Amealing ^:

Extension ^:

95oC, 2min

95oC, 1 min‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑

48oC, 1min‑‑‑‑‑‑X 30 cycles‑‑‑

68oC, 2min‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑

Per product rfcA's length was

‑1.7kb

(Fig. 7).

I M

Fig. 7. RfcA

(0.8%

agarose, 100V,

30min).

^{Lane M: i EcoT} marker, lane 1:RfcA.

3.4 Construct the plasmids

3.4.1 BPreaction

Transfer P‑BKatE into the vector PDONR P1

‑P5r.

Trahsfer Hup‑terminater into the vector PDONR P5‑P2.

The components of reaction:

Fragment PDONR ^vector TE buffer

P‑BKatE 1 p1

(‑80ng)

1 p1 6Lll

Hup‑teminator 1 p1

(‑80ng)

^{1 p1 6p1}

The PDONR: :Hup‑terminater, PDONR::P‑BKatE by BP reacted ^were checked by PCR with GOTaq. Fig. 8.

Template: PDONR:P‑BKatE

r

Primer:

PDONR:P‑BKatE‑‑ Hup‑f and BKatE‑r Cycling step:

Pre‑denaturation :

D enaturation ^:

Annealing :

Extension:

95oC, 2min

95oC, 1min‑‑‑‑‑‑‑‑‑‑‑‑‑‑

55oC, 1min‑‑i‑X 30 cycles‑‑‑‑‑

68oC, 2min20sec‑‑‑‑‑‑.‑‑‑‑‑‑‑‑‑‑‑‑

Template: PDONR:Hup‑terminator

Forward primer: Tem‑f

Forward primer: Tem‑r

Cycling step:

Pre‑denaturation :

D enaturation ^:

Amealing ^:

Extension :

95oC, 2min

95oC, 1 min‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑

60oC, lmin‑‑‑‑‑‑X 30 cycles‑‑‑‑‑

68oC, 1 5sec‑‑‑‑‑‑‑‑‑‑‑‑‑‑

1 2 3 4 5 6 7 8 9 M

Fig, 8a. PDONR::P‑BKatE

(0.8%

agarose, 100V,

30min).

^{Lane M: i EcoT} marker, lane 4‑6: PDONR: :P‑BKatE.

Fig. 8b, PDONR:Hup‑terminator

(2%

agarose, 100V,

30min).

^{Lane M: lOObp}

marker, lane 1:PDONR:Hup‑terminator.

3.4.2 LR reaction

Ligated Hup‑terminater and P‑BKatE by LR reaction.

The components of reaction:

PDONR PGUOO 1 PDONR: TE buffer

Hup‑terminator

pBCATOO1

1pl(‑10hol) 1p1(‑20ho1) 1pl(‑10hol)

^5p1

The plasmids, pBCATOOl ^were checked by PCR with GOTaq. Fig. 9.

M l 2 3 4 5 6 7 8

Fig. 9. pBCATOO1

(0.8%

agarose, 100V,

30min).

^{Lane M: i EcoT} marker, lane 4‑6:

pBCATOO1.

3.5 Analyze the sequences

The plasmid pBCATOOl is showed below

(Fig. 10):

...aacgcgggttTTcgcagaaaCatgCgCtagfaTcLTftgatgacaacatgg actaagcaaaagtgcttgtcccctgacccaAGAAGGatgcttt4TG‑..

".ccttctgctcgtagcgattacttcgagcattaCtgaCgaCaaagaeCC(

gaccgagatggtcggggtctttttgttgtggTgCtgtgaCgtgttgtCea

^‑.

Fig. 10. The map of pBCATOOl construction using the Gateway system. P: hup

promoter; T: hup teminator; katE: B. subtilis heme‑dependent _catalase; ^Spr:

spectinomycin resistance; repB:

Bljidobacterium

replication initiation; ColEl ori:

colicine El

origin

^of replication; attB1, attB2, attB5: Gateway system sites; bold:

palindromes; italic: proposed ‑35 and ‑10; uppercase: proposed RBS; uppercase italic:

the initiation codon̲

3.5.1 Hup‑promoter

The Hup‑promoter sequence is showed below:

tagatgtgaaaacccttataaaacgcgggt

‑35

gaaacatgcgctaa;i;*gatgacaacatggactaagc

‑10 aaaagtgcttgtcccctgacccaagaaggatgcttt

RBS

The proposed promoter

(‑35,‑10)

^{and a ribosomal binding sequence were boxed}

and underlined with ^a double line, respectively.

3.5.2 Hup‑terminator

The Hup‑terminator sequence is showed below:

ccttctgctcgtagcgattacttcgagcattactgacgacaaagaccccgaccgatggtcggggtctttttgttgtggt

gctgtgacgtgttgtccaaccgtattattccggactagttcagcg

The _{regions of} dayd symmetry ^are indicated by horizontal arrows and the following T‑rich sequence is showed by a double line.

3.5.3 Catalasegene

Comparison of the B‑KatE

(Heme‑catalase)

of B. subtilis GTCO1672 with B.

subtilis subsp. subtilis ^st7: 168 indicated no mismatched.

3.6 The catalase activity inE. coli UM255

Escherichia coli UM255

bro

leu rpsL hsdM hsdR endl lacy katG2 katE::Tn10

recA)

was used ^{as a} host strain for the gene cloing of catalase. Transformated E. coli

uM255 with pBCATOO1. Detected activity of catalase by adding ^{H202 tO the Plate}

colonies. See the SDS‑PAGE in the Fig. 12.

1n Fig. 1 1, catalase activity^{was observed} in E. colt UM255.

Fig. 1 1. The presence ofE. colt UM255

bBCATOO1)

catalase activity leads ^to bubble formation resulting &om the transformation of H202 ^tO H20 and 02.

3.7 The catalase activity in B. Longum 105‑A

The catalase activity ^was observed ^{in E.} colt UM255, then, detected the catalase

activity in B. longum lO5‑A.

3.7. 1 PBCATOO I

(Heme‑catalase)

activity

The gene encoding the Heme‑dependent catalase

(pBCATOO1)

of B. subtilis ^was introduced into B. longum 105‑A. Transformated B. Longum lO5‑A was grown in MRS medium. Catalase activity ^was checked by adding H202. No bubble was

observed. Then, transformated B. longum 105‑A was grown in MRS medium added Hemin

(10LtM).

^Checked the catalase activity with the method ^2.7.1.

We then determined the catalase protein in A colt UM255 and B. longum 105‑A

cultures ^on SDS‑PAGE. In the fractions, ^one ‑77 kDa migrating band was detected and present in E. colt

UM255bBCATOO1)

^extract, suggesting that it could

UM255 with pBCATOO1. Detected activity of catalase by adding H202 tO the plate

colonies. See the SDS‑PAGE in the Fig. 12.

In Fig. ll, catalase activity ^was observed in E. coli UM255.

T‑I I

Fig. 1 1. The presence ofE. coli UM255

bBCATOO1)

catalase activity leads ^to bubble formation resulting from the transformation ofH202 ^tO H20 and 02.

3.7 The catalase activity in B. longum 105‑A

The catalase activity ^was observed in E. coli UM255, then, detected the catalase

activity in B. longum 105‑A.

3.7. 1 pBCATOO1

(Heme‑catalase)

^activity

The gene encoding the Heme‑dependent catalase

bBCATOO1)

of B. subtilis ^was introduced into B. longum 105‑A. Transformated B. longum 105‑A was grown in MRS medium. Catalase activity ^was checked by adding H202. No bubble was

observed. Then, transformated B. longum 105‑A was grown in MRS medium added Hemin

(10pM).

^Checked the catalase activity with the method 2.7.1.

We then determined the catalase protein in E. coli UM255 and B. longum 105‑A

cultures ^on SDS‑PAGE. In the fractions, ^one ‑77 kDa migrating band was detected and present in E. coli

UM255bBCATOO1)

^extract, suggesting that it could

correspond ^to BKatE. No corresponding band was detected in B. longum 105‑A. The protein ofcatalase ^was checked by SDS‑PAGE

(Fig. 12).

Fig. 12. Catalase in E. coli UM255 and B. longum lO5‑A on SDS‑PAGE

(10%

running gel, 4% stacking

gel).

^BKatE

(Heme‑catalase

^{from B.}

subtilis)

^{in E. coli}

UM255 ^was visible. BKatE in B. longum 105‑A was invisible. Lane M: Protein MW Maker

(Daiichi),

lane 2: E. coli UM255

bBCATOO1),

^lane 4: B. longum 105‑A, lane 6:

B. longum 105‑A

bBCATOO1).

In Fig. 13, catalase activity ^was observed in B. longum 105‑A.

a)

^{B. longum 105‑A}

bKKT427)

B, longum lO5‑A

O)BCATOO1)

r

=

l3

‑

0

LI

A En

E

〜

=

tJ

>

:i

_O

<

0 u)

〜

7;

1I

ql

O 0

A B C

Fig. l3. Comparative assay of KatE activity. The degradation of H202 ^Was measured in

crude protein ^extracts. A: B. longum 105‑A harboring pBCATOO1

(hup promc.ter);

^{B: B,}

longum 105‑A harboring pBCATOO2

kap _promoter);

^{C: B. longum 105‑A harboring}

pBCATOO3

(BkatE

native

promoter).

^{Error bars correspond to} the standard ^errors of the

means.

The catalase activity data ^see below:

Table S2. The catalase activities of crude ^extract.

(U/mg)

B. longum B. longum B. longum

Hemina ^. 105‑A

(wild

^{105‑A 105‑A}

type) bKKT427) bBCATOO

¹

)

nd nd 39

nd nd nd

iEgd:e:r:aidi1;;:eenrco:efg e(c;e;nsdilttnioa;e.d?ilBE6;fg??oT

^{em in in/ m edium '}

3.8 Short‑term H202 exposure OfB. longum 105‑A

The production of BKatE by B. longum 105‑A effect under oxidative ^stress

conditions ^was evaluated by oxidative stress induced by adding H202.

B. longum 105‑A

(pBCATOO1)

^{was incubated for 1 h in MRS} medium in the presence of 4.4 mM H202 concentrations ^to determine discriminating conditions that would allow the evaluation of the effect of BKatE ^on the survival of exponentially growing cells

(OD660‑0.6)

^or stationary‑phasep cells

(OD660‑1.0).

^The production of BKatE in B. longum 105‑A conferred improved survival ^{rates were} better H202

resistance than the control strain. The survival of exponentially growing cells and stationary‑phase cells in response ^to 4.4 mM H202 Was increased 120‑fold

(The

^data

see

below).

CFU/mL and survival ^rates after H202 exposure for 1 h

Gro wth phase

Strain

CFU/mL after H202 exposure

Survival

OmMH202 4.4mMH202 rateSa

Survival rate increasesb Exponential

0D660 0.6

S tationary OD660 1.0

B. longum 105‑A

bKKT42 7)

B. longum 105‑A

(PBCATOO

¹

)

B. longum 105‑A

(pKKT42 7)

B. longum 105‑A

(PBCATOO

¹

)

7.00x 108

(iO.34)

8.00x 108

(iO.16)

1.68x 109

(j=0.22)

1.56x 109

(iO.34)

1.20x 105

(iO.20)

1.64x 107

(iO.26)

2.lox 106

(iO.40)

2.00x 108

(j=0. 14)

0.00017

0.021 120 fold

0.0013

0.13 103 fold

a Survival _rates were generated by comparing the colony ^counts for 0 versus 4.4 mM H202 exposure for 1 h _at 37oC.

b survival _rate increases were compared for B. longum 105‑A

(pBCATOO1)

and B.

longum 1 05‑A

bKKT427).

*Data _are the ^{means i} standard deviations of three independent experiments.

3.9 Long‑term 'with aerated cultures ofB. longum 105‑A

B. longum is known to accumulate H202 during its growth in the presence of 02.

We therefore also compared the long‑term survival ofB. longum 105‑A

(pBCATOO1)

during aerated growth. Since BKatE requires exogenous hemin for activity, these

aerated cultures supplemented with hemin will lead ^to respiratory metabolism.

3.9.1 0D

The OD values under aerobic and anaerobic condition ^were assayed.

0 1 0 20 30 40 50

Time (h)

Fig. 14. Cultured B. longum 105‑A

bKKT427

^or

pBCATOO1)

^{under aerobic or}

anaerobic conditions. Growth

(OD660)

Of the cultured B. longum 1 05‑A. Closed circle

(.),

^{B. longum 105‑A}

bKKT427)

cultured under aerobic conditions; open circle

(o),

B. longum 1 05‑A

bBCATOO1)

^{cultured under aerobic} conditions; closed triangle

(A),

B. longum 1 05‑A

bKKT427)

cultured under

ahaerobic

conditions; closed square

(‑),

B. longum 1 05‑A

(pKKT427)

cultured using exogenously added catalase

(3000

^U/mL

medium)

^{under aerobic} conditions; open square

(u),

^{B. longum 105‑A}

(pKKT427)

cultured using exogenously added catalase

(100

^U/mL

medium)

^{under aerobic}

conditions. The results presented correspond ^to the averages of three different assays.

Error bars correspond ^to the standard ^errors of the ^mean value.

3.9,2 CFU

The CFU under under aerobic and anaerobic condition ^were assayed.

9

A

25E !g5

7

5

3

10 20

Time (h)

30 40 50

Fig. 15. Cultured B. longum lO5‑A

bKKT427

^or

pBCATOO1)

^{under aerobic or}

anaerobic conditions. Counts of B, longum 105‑A cultured under aerobic conditions.

Closed circle

(.),

^{B. longum 105‑A}

O)KKT427)

cultured under aerobic conditions; open circle

(o),

^{B. longum 105‑A}

bBCATOO1)

cultured under aerobic conditions; closed square

((),

^{B. longum 105‑A}

(pKKT427)

cultured using exogenously added catalase

(3000

^U/mL

medium)

^{under aerobic} conditions; open square

(u),

^{B. longum lO5‑A}

bKKT427)

cultured using exogenously added catalase

(100

^U/mL

medium)

^under

aerobic conditions. The results presented correspond ^to the averages of three different assays. Error bars correspond ^to the standard ^errors of the ^mean value.

3.9.3 LIVE/DEAD

The live and dead cells under aerobic and anaerobic condition ^were assayed using flow cytometry.

8

A

W J

? E

Bi7 jP

V

6

10 20 30 40 50

Time (h)

Fig. 16. Cultured B. longum 105‑A

bKKT427

^or

pBCATOO1)

^{under aerobic or}

anaerobic conditions. L/D assay of B, longum 105‑A cultured under aerobic conditions. Closed circle

(.),

^{BJongum 105‑A}

bKKT427)

cultured under aerobic conditions; open circle

(o),

^{B. longum 105‑A}

(pBCATOO1)

cultured under aerobic conditions; The results presented correspond ^to the averages of three different assays.

Error bars correspond ^to the standard ^errors of the ^mean value.

3.9.4 H202 accumulation

The H202 accumulation under aerobic and anaerobic condition ^were assayed.

0.12

A

2 E

〜

0

L5

E o

O

oNo

〜

I

o

.06 .04 .02 0

1 0 20 30 40 50

Time (h)

Fig. 17. Cultured B. longum 105‑A

(pKKT427

^or

pBCATOO1)

^{under aerobic or}

anaerobic conditions. H202 accumulation by B. longum 1 05‑A cultured under aerobic conditions. Closed circle

(+),

^{B. longum, 105‑A}

(pKKT427)

cultured under aerobic conditions; open circle

(p),

^{B. longum 105‑A}

(pBCATOO1)

cultured under aerobic conditions; The results presented correspond ^to the averages of three different assays.

Error bars correspond ^to the standard ^errors of the ^mean value.

ドキュメント内 Acquired Tolerance to Oxygen Stress in Bifidobacterium longum 105-A by Heterologous Expression of Catalase Gene (ページ 56-75)