Acquired Tolerance to Oxygen Stress in Bifidobacterium longum 105-A by Heterologous Expression of Catalase Gene

Title

Acquired Tolerance to Oxygen Stress in Bifidobacterium longum

105-A by Heterologous Expression of Catalase Gene( 本文

（FULLTEXT) )

Author(s)

賀, 建龍

Report No.(Doctoral

Degree)

博士(農学) 甲第583号

Issue Date

2012-03-13

Type

博士論文

Version

publisher

URL

http://hdl.handle.net/20.500.12099/42969

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

Contents

1. Introduction---I--- 1 1. 1

_{Bljidobacterium---}

1 1.1.1 Description---1 1.1.2 History---4 1.1.3 Species---'---I---5 1.1.4 Ecology---6 1.2 Peroxidase---9 1.2.1 Description---i--i---9 1.2.2 GPX---i---10 1.2.3 Catalase---J---12 1.2.3. 1 Molecular _{mechanism---1} 3 1.2.3.2 Cellular _role---14

1.2.3.3 Distribution _{among organisms---1} 5 1.3 Research the response of the

Bljidobacterium

to Oxygen---1 7 2. Materials _and Methods---20

2. 1 Media _and Buffer---i---20

2. 1. 1 Luria Broth _media---20

2.1.2 MRS _{media---,---20}

2.1.3 TE buffer---I---21

2.1.4

_PEG---:---21

2. 1.5 PBS buffer--I---21

2.2 Isolate DNA from _{microorganisms---2} 1 2.3 Extract _plasmid---2 1 2.3.1 With

_QIA

_{prep spin Mini prep} kit---21

2.3.2 With 2-propano1---22 2.3.2. 1 The _{reagent---.---22} 2.3.2.1. 1 Solution I---22 2.3.2. 1.2 Solution II---22 2.3.2. 1.3 Solution III---22 2.3.2.2 Protocol---22 2.4 Purify DNA---23

2.4.1 With Nucleo Spin Extract II kit---24

2.4.2 With Ethano1---24

2.4.2. 1 I The _reagent---24

2.4.3 With PEG---25

2.4.3. 1 The _reagent---25

2.4.3.2 Protocol---25

2.5 Preparation _{of competent cell---25}

2.5.1 Electrocompetent _{cell of BiBdobacterium---25}

2.5. 1. 1 The _reagent---25

2.5.1.2 Protocol---26

2.5.2 Chemically _{competent cell of} E.boli---26

2.5.2. 1 The _reagent---26

2.5.2.2 Protocol---27

2.6 SDS-PAGE---i---27

2.6.1 The _reagent---27

2.6.2

_{Protocol---i-I--29}

2.7 Detection _{activity of catalase---32}

2.7.1 Add H202 detect _activity.f

catalas;---i---32

2.7.1.1 The

_{the.,y-;---i---32}

2.7. 1.2 Protocol---.---32 2.7.2 Assay _{ofcatalase---32} 2.7.2. 1 The _reagent---33 2.7.2.2 Protocol---33 2.8 Short-term H202 _{exposure---34} 2.8.1 The _reagent---34 2.8.2 Protocol---34

2.9 Long-term _{with aerated cultures---35}

2.9.1 The _reagent---35

2.9.2 Protocol---I""_"___"__"______"__""_MM___M""__"Mum""__"__35 2. 1 0 PCR---3 5 2.10.1 With _po1ymerase KOD-PLUS---i---35

2.10.2 With _po1ymerase GO Taq---35

2. 1 1 DNA _extraction from _qgarose

gels---36

2.12 Digestion _{with restriction enzyme---T---36}

2. 12. 1 The _restricti.n

enzyme---J-i---36

2. 12.2 Protocol---36 2. 1 3 DNA ligation---I---36 2. 1 4 Transformation---36 2. 14.1 Chemical transformation---37 2. 14. 1. 1 The _reagent---37 2. 14. 1.2 Protocol---37

2. 14.2 Electroporation---38 2. 14.2. 1 The

reagent---;---38

2. 14.2.2 Protocol---i--38 2. 1 5 Gateway _system---3 8 2.16 Flow _{cytometry---39} 2. 16.1 The _{reagent---.---39} 2. 1 6.2 The instrument---39

2. 1 7 Isolate RNA from _{microorganisms---I---39}

2. 1 8 RT-PCR---39 2.19 _qRT-PCR---39 2.19.1 The _reagent---39 2. 1 9.2 The instrument---39 2.20 Assay _{of H202---40} 2.20. 1 The

reagent---i---40

2.20.2 Protocol---40 2.2 1 Site-Directed Mutagenesis---40

2.22 The _{strains, the plasmids and primers---42}

3. Results---44

3. 1 Isolate DNA &om _{microorganisms---i---44}

3.2 Get the PCR _product---45

3.2.1 Design _{the prim'ers---45}

3.2.2 PCR---45

3.2.2. 1 PCR _{of Hup-promoter---45}

3.2.2.2 PCR

_{ofB-KatE---i_"MUM"___"__47}

3.2.2.3 PCR _{of Hup-terminator---48}

3.2.2.4 Overlap PCR---49

3.3 Construct destination _vector---50

3.3.1 Digest _pKKT427---50

3.3.2 Construct destination vector pGUOO1---51

3.4 Construct _{the plasmids---53}

3.4. 1 BP _{reaction---53}

3.4.2 LR

_{,eacti.n---_M"_"_"____""""q_____"_:MM____56}

3.5 Analyze _{the sequences---57}

3.5.1 Hup-promoter---58

3.5.2 Hup-terminator---58

3.6 The _{catalase activity in E. coli UM255---58}

3.7 The _{catalase activity in B. longum} 1 05-A--.---59

3.7.1

_pBCATOO1

_{(Heme-catalase)}

_{activity---59}

3.8 Short-term H202 _{exposure OfB.longum} 105-A---63

3.9 Long-term _{with aerated cultures ofB.longum} 105-A-i---64

3.9. 1 0D---64

3.9.2 CFU---65

3.9.3 LIVE/DEAD---66

3.9.4 H202 _{accumulation---67}

4. Discussion---68

4. 1 Protect

_{Bljidobacterium}

from _oxidative stress by expression of catalase---68

4.2 Improvement _{of catalase expression} level---7 1 4.2.1 Transcription---71

4.2.2 Translation---75

5. Conclusion---85

6. References---89

Intro duction

1.1

_{Bljidobacterium}

1.1.1- Description

At birth,_{the gastrointestinal} tract _{is sterile and incapable of} digesting food. Within

hours, bacteria ingested during the birthing _{process rapidly colonize} the _{gut. The}

gastrointestinal tract soon _{contains about} 1 0 times as _many bacteria as _there are _cells

in the body. Hundreds _{of species} are _present, many of which are _{uncultivable and}

remain unidentified. It is these bacteria that are responsible for priming the

gastrointestinal immune system. This gut flora includes 100 trillion bacteria, some

three pounds, which are intimately linked to the bodyTs natural defense system.

(18)

probiotics are deflned as live microbial food ingredients that benefit human health.

Most _probiotics fall into the _{group of organisms} known as lactic acid-producing

bacteria _and are _{normally consumed} in the form _{of yogurt,} fermented milks or _other

fermented foods. The _{concept ofprobiotics} arose atthe turn of the 20th century from

a hypothesis flrSt Proposed by. Noble Prize winning Russian scientist Elie

Metchnikoff, _{who suggest} that the long, healthy life of Bulgarian peasants resulted from _{their consumption of fermented milk products.} He believed _{that when consumed,} the fermenting Bacillus

_{(Lactobacillus)}

_positively influenced _{the micro flora of the} colon, decreasing toxic microbial activities. The historical association of probiotics

with fermented dairy products, stilltrue today, stems from these early observations.

Investigations _{in the probiotics} field during _{the past several} decades, however, have

expanded beyond bacteria isolated from fermented dairy products to those of

Possible health _{effects of probiotics}

Intestinal

_eHects

･ _Relieve

effects, promote recovery from diarrhea

(rotavirus,

travelers' and

antibioticinduced)

･ produce

lactase, alleviate symptoms of lactose intolerance and malabsorption

･ Relieve

constipation

･ _Treat

colitis

Immune _system

_eHects

･ _Enhance

specific and nonspeciflC immune response

･

Inhibit pathogen growth and translocation

･ _Stimulate

gastrointestinal immunity

･ _Reduce

chance of infection from common _pathogens

(Salmonella,Shigella)

Other

eHects

･ Reduce risk of certain _cancers

(colon,bladder)

･ _Detoxify

carcinogens

･ Suppress _tumors ･ Lower'

serum _{cholesterol concentrations}

･ _Reduce blood

pressure in hypertensives

･ _Treat food

allergies

･ synthesize

nutrients

(folic

acid, niacin, riboflavin, vitamins B6 & B

12)

･ _Increase

nutrient bioavailability

･ _Improve

urogenital health

･ Optimize

effects of vaccines

(e.g.

rotavirus vaccine, typhoid fever

_vaccine)

There is some debate _{about whether} or not yogurt starter bacteria should be

Streptotoccus thermophilus are _used to ferment _{milk and} turn _{it into yogurt. But these} cultures are not very resistant to conditions in the stomach and small intestine and

generally do not reach the gastrointestinal tract _{in very high numbers.} 'Therefore, they cannot mediate some _{probiotic effects. But} these starter bacteria have been shown to

improve lactose digestion in people lacking lactase _and have demonstrated some

itnmunity _{enhancing effects. For these} reasons, they are _{often considered} 'probiotic'.

Most _{gastrointestinal organisms} are _relatively benign. Some are _potentially more

pathogenic; however, many are _actually beneficial; it is these beneBcial _organisms

that have _{attracted attention as.possible probiotics.} The table below lists some

suggested health benefits of consuming probiotics. Those that have signiflCant

research to back up the claims are discussed in more depth later in this article.

(79)

Bljidobacterium,

one type _{of probiotics,} are a _{natural part of the bacterial} flora in

the human body _and have a _symbiotic bacteria-host _{relationship with} humans. B.

longum _{promotes good} digestion, boosts the immune system, and produces lactic and

acetic acid that controls intestinal pH. These bacteria also inhibit the growth of

Candida _{albicans, E. coli}

_(9),

_{and other} bacteria that have more _{pathogenic qualities}

than

Bljidobacterium. Bljidobacterium

are _normal inhabitants _of the human _and

animal colon. Newborns, especially those that are breast-fed, are _{colonized with}

bifldobacteria _within days _{after birth.}

_{Bljidobacterium}

were first isolated &om the

feces _{of breast-fed} infants. The _{population of these} bacteria in the _{colon appears}

relatively stable until advanced age, when itseems to decline. They are _{saccharolytic}

organisms that produce acetic and lactic acids without generation of CO2, except during degradation _{of gluconate.} They are _{also classified} as lactic _acid bacteria

Bindobacterium www. _{sciencephoto.} com

1.1.2 History

Bljidobacterium

was Brst isolated as a bacteria in France in 1 899. Itwas found in a

healthy breast-fed baby, but the bacteria did _not come from the breast _milk. Itcame

B-om _{something the baby} had _consumed. The first name

bljidobacterium

was _called

Bacillus

bljidus

communis. There are 30 different _{types of}

bljidobaclerium

_species

that exist. When this was known, in 1960,

bljidobacterium

was _accepted as its own

1.1.3 Species

The table below _{lists the species ofbifidobacteria.}

Bljidobacterium

angulatum

Bljidobacterium

animalis

Bljidobacterium

asteroids

Bljidobacterium

bljidum

Bljidobacterium

boum

Bljidobacterium

breve

Bljidobacterium

catenulatum

Bljidobacterium

choerinum

Bljidobacteriumco7yneforme

Bljidobacterium

cuniculi

Bljidobacterium

dentium

Bljidobacterium

gallicum

Bljido

bacterium _gallinarum

Bljidobacterium

indicum

Bljidobacterium

longum

Bljidobacterium

magnum

Bljidobacterium

mefyCicum

Bljidobacterium

sp

Bljidobacterium

pseudolongum

Bljidobacterium

_psych;aeroph

ilum

Bljidobacterium

pullorum

Bljidobacterium

ruminantium

Bljidobacterium

saeculare

Bljidobacterium

scardovii

Bljidobacterium

simiae

Bljidobacterium

subtile

Bljidobacterium

thermacidophilum

Bljidobacterium

thermophilum

Bljidobacterium

urinalis

Bljidobacterium

magnum

Bljidobacterium

m _e7yCicum

Bljidobacterium

minimum

Bljidobacterium

pseudocatenulatum

Bljidobacterium

minimum

1.1.4 Ecology

While

_{Bljidobacterium}

_infantis,

B. brevi, _and B. longum are _{the largest group of}

bacteria _{in the intestine of infants,}

Bljidobacteria

are _said to be _only the 3rd or 4th

largest _{group in adults}

_(and

_{comprise only} 3-6% _{of adult} fecal

_nora).

The _number-of

Bljidobacteria

actually decline in the human body with age. In infants who are

breast-fed,

_{Bljidobacteria}

_constitute

ab6ut

90% of their intestinal bacteria;- however, this number is lower in

_bottle-fed

infants. When breast-fed infantsT diets are _changed

to cows _{milk and solid} food,

Bljidobacteria

are

joined

by rising _{numbers of other}

bacteria found ihthe human body such as Bacteroides _and Streptococci lactobacilli.

The lower _number

,ofBljidobacteria

in formula-fed babies _{might account} for a higher

risk _of diarrhea _{and allergies} that is usually associated with babies who aren't breast-fed; _{in addition,} because

_{Bljidobacteria}

_{produces lactic acid instead of gas}

(like

E.

colt),

infants and people in general with more

_{Bljidobacteria}

than other bacteria _will have less _{gas and} digestive _problems. There is also a _significant

difference in the incidence of antibiotic-associated diarrhea in the children receiving

probiotic-supplemented

(inriched

with

Bljidobacterium)

formula

(16%)

than nonsupplemented formula

(31%). (14,

68, 88,

90)

Tissier described B.

_bljidus

_{in the stools of breast-fed} infants, itwas _recognized

that the organisms became _{predominant in the stools} between days _{4 and 7 after} birth.

Although _{L. acidophilus} was the most numerous lorganism in the stools of bottle-fed

infants, bifidobacteria were _also found in _{smaller numbers.} It was believed _that

bifidobacteria were _present in small _numbers in _{stools of adults,} but _they were

difficult to isolate because _{of the lack of} a _{suitable culture medium} or _{method of}

reducing oxygen tension. For the next half century little progress was _made in

interest in the occurrence _and distribution _{of bifidobacteria} was _revived. Mata et a1.

reported the average figures for the incidence of bifldobacteria in the feces of breast-fed infants to be _{approximately} loll, in weanlings 1010, _and in adults 101

organ.isms per g

(wet weight).

The figures expressed by these authors are in general

agreement with those expressed earlier by Gyllenberg and

Roin;,

Smith and Crabb, Zubrzycki _and Spaulding,

_Weijers

_and van de Kamer, _and Werner. They are

somewhat higher than those reported by Kalser et a1. Werner and Seeliger, Mossel,

and Gorbach et a1. In a _{study ofintestina1} flora in a _rural area _ofGuatemala,

_Mata

_and

urrutia _reported that bill-dobacteria appeared on the flrSt day of life in only a few

infants. One-third _{of the babies} they studied had these bacteria on _{the second} day _of

life. By the end of the flrSt Week all infants had them in concentrations _ranging &om 101 to _{loll organisms per g of feces.} These _{workers also reported} that Bacteroides and Veillonellae were not frequently found in the stools of breast-fed neonates,

although their concentration ranged from 108 to _{1011 when} found. When 12 breast-fed infants were _studied throughout _{the flrSt year Of life, bifldobacteria continued} to be

the most numerous bacterium, _amounting to 1010 to 1011 _{organisms per g of feces.}

With food _{supplementation} the _{anaerobic gram-negative} bacilli became more

numerous _{and eventually outnumbered other} bacterial _groups. Mata _and Urrutia

summarized their data by stating that nearly 100% _{of all bacteria cultured} from the

stools of breast-fed infants were bifidobacteria. During _{weaning there} was a decrease

by 1 log _and a _{proliferation of Bacteroides. In adults, Bacteroides outnumbered all}

other groups. Although this study was a _{valuable aid} in _estimating the relative

occurrence _{of various groups of} intestinal_{organisms and their variation with age and}

other factors, no _attempt was _{made at} a hef _{classification of members of the genus}

Bifidobacterium. These _organisms were _{simply referred} to as the bifidobacteria _group.

classiflCation schemes. Miller reported on the fecal flora of seven _{eskimo children.}

He _{reported B. adolescentis in concentration of 108} to _{loll per g in all the specimens}

studied and noted that in one case _{members of the genus} bifidobactera _exceeded those

of the bacteroides group. In a _{study of the} intestinal flora _{of adults,} Moore et a1.

reported that in some cases _{members of bifldobactera} outnumbered bacteroides, thus

confirming the observations of some earlier workers on this point. Significant

differences in incidence _{of bifidobacteria among} individuals _{and possibly among}

certain restricted populations are _evident. An investigation of variation in the

incidence _{ofbifidobacteria among newborns} was _carried out in our laboratory. In the

course _{of investigations,} we _noted fewer bifidobacteria in newborns in a large, urban

universityl hospital than in a _suburban hospital in the same area. In the _past,

bifldobacteria _{could readily} be _cultured from the _stools

6f

breast-fed infants _at the Hospital _{of the University of Pennsylvania.} Recently, difficulty was _experienced in

culturing these organisms from the stools of infants in the nursery. Gram-stained spreads of feces of

breast-fed

infants, 3 to 4 days old, were _examined. Of 61

breast-fed infants

_(14,

35, 36, 37, 38,

₆₆₎

Bljidobacteria

as _well as _{other beneficial} bacteria can be found in fermented dairy

foods, _{especially yogurt.} Eating _substances rich _with these _probiotics is a _{sort of}

home _remedy for diarrhea, _{vaginitis, and}

_yeast infections because it promotes the growth of these as _opposed to _other bacteria. B. infantis has been _proven to

dramatically _reduce imitable bowel _sydrome

_(IBS)

_{in patients and if given alone} can

StzlePtOCOCCUS

S. thern70PJLj]usATCC l9258 S.8Lljb,ATCC437 65 1.2 Peroxidase 1.2.1 Description IF

Gran(-)

OeJ20CLmuS _{OeL7j JCM6} I 25

Lactococcus

Genealogical tree

Peroxidases

(EC

number

1.ll.1.A)

are a large family _{of enzymes} that typically

catalyze a _{reaction of the form:}

ROORr +

electron donor

(2 e-)

+ 2H' - Roll + R'OI1

For _{many of these enzymes the optimal substrate} is hydrogen _{peroxide, but others}

are more _{active with organic} hydroperc.xides _such as lipid peroxides. Peroxidases can

contain a heme co factor in _{their active sites,} or _{redox-active cysteine} or

selenocysteine residues.

Peroxidase can be _used for treatment _{of industrial} waste waters. For example, phenols, which are important _pollutants, can be _removed by _{enzyme-catalyzed}

radicals, which participate in reactions where polymers and oligomers are _produced

that are less toxic than phenols.

Furthermore, _peroxidases can be an _{alternative option of} a _{number of} harsh

chemicals, eliminating harsh reaction conditions. There are _{many investigations}

about

the use of peroxidase in many manufacturing processes like adhesives, computer

chips, carparts, and linings of drums and cans.

(10,

13, 20, 34, 43, 63,

₇₄₎

The table below lists the types ofperoxidases.

I _Glutathione peroxidase

(GPX)

･ Haloperoxidase ･ Myeloperoxidase

(MPO)

･

Catalase(Rat)

･ Hemoprotein ･ Peroxide I _{Peroxiredoxin} I Animal heme-dependent peroxidases ･ Thyroid peroxidase I Vanadium bromoperoxidase ･ Lactoperoxidase 1.2.2 GPX

Glutathione _peroxidase

_{(GPX) (EC 1.ll.1.9)}

_{is the general} name

of an _enzyme

family _{with peroxidase activity whose main} biological _{role is} to _{protect the organism} from _oxidative damage. The biochemica1

_functioh

_{of glutathione peroxidase} is to reduce lipid hydroperoxides to their corresponding alcohols and to reduce free

hydrogen _peroxide to water.

(2,

3, 6, 34,

₅₄₎

GPX family _{is well studied in eukaryote, especially in animals, and} act a key _role

in H202

_scavenging.

In the case _{of bacteria, very} limited information _about GPX were

available. Itwas _reported in few _{of species,} Neisseria _meningitidis

(Aho

E. L. et _a1.

1995)

Streptococcuspyogenes

(King

K. Y. et _aL.

2000)

and Eschericha colt

(Arenas

F.

A. et al

2010).

It is not commonly accepted that GPX _plays an important _role in

bacteria, _especially in anaerobic bacteria. No _orthologue to _these 3 bacterial GPX

(also

Eukaryotic

_GPX)

has been found in the bifidobacterial _genome. Glutathione synthesize pathway has not been found in bifidobacteria

(figure

see

below). (79, 80)

1.2.3 Catalase

Catalase is a common _enzyme found in nearly _{all living organisms which} are

exposed to oxygen, where it functions to _catalyze the decomposition _{of hydrogen}

peroxide to water and oxygen. Catalase has one _{of the highest} turnover _{numbers of all}

enzymes; one _{molecule of catalase} can convert millions of molecules of hydrogen

peroxide to water and oxygen per second.

(1,

5, 10, 20, 22, 23, 45, 50, 63,

72)

Catalase isa tetramer _{of four polypeptide chains, each} over 500 _{amino acids} long. It contains four porphyrin heme

_(iron)

_{groups that allow the enzyme to react with the} hydrogen _peroxide. The _{optimum pH} for _{catalase is approximately 7, while the}

optimum temperature varies by species.

Catalase

_(EC

1.1

_1.1.6)

_catalyzes disproportioning _ofH202 tO H20 _and 02 _and thus protects the cells against the oxidative effect of H202. This enzyme is present in all aerobes and many aerotolerant anaerobes. Two types of phylogenetically remote

heme _catalases are known: _{monofunctional catalases and} bifunctional _catalases

peroxidases, which

for

the catalase activity use H202

(Km

- 2.5 6.5

mM)

as an

electron donor and for the peroxidase activity

(Km

- 0.2 0.7

mM)

use _various

organic compounds

byroga11o1,

diaminobenzidine, dimethoxybenzidine, dianizidine, NADH, NADPH,

_etc.).

Monofunctional _catalases are found in all three empires _of

living nature, _whereas the distribution _{of bifunctional} heme _catalases is limited

_(with.

rare

_exceptions)

to bacteria _{and archaea.}

Unlike mono- _and dimeric bifunctional _{catalases-peroxidases, monofunctional}

catalases are _mainly tetrameric _{proteins characterized} by higher temperature _stability,

wide pH optimum

(5.5-16.5),

and lack of inactivation with ethan.I/ch1.r.f.rm. However, 3-amino-1

In addition to heme _catalases, there are Mn _{catalases with} a _{unique primary} structure and resistance to N3-, _which have been found in some facultative _{anaerobes such} as

the lactic acid bacterium Lactobacillus _{plantarum and} the hyperthermophilic _archaeon

Pyrobaculum _{calidifontis.}

Catalase was first noticed as a _substance in 1811 _{when Louis Jacques Th6nard,}

who discovered H202

(hydrogen peroxide),

suggested that its breakdown is caused by

a _substance. In 1900, Oscar Loew was the firstto _give it the name _{catalase, and} found

its presence in _{many plants and animals.} In 1937 _catalase &om beef liver was

crystallised by James B. Sumner and the molecular weight worked out in 1938. In

1969 _{the amino acid sequence of bovine catalase} was _{worked out.} Then in 1981, _the

3D structure of the protein was

revealedi

1.2.3.1 Molecular _mechanism

The _{reaction of catalase} in the decomposition of hydrogen peroxide is:

2H202 -+ 2H20+02

While _{the complete mechanism of catalase} is _{not currently} known, _{the reaction} is believed to occur in two _stages:

H202 +

_Fe(III)-E.)

H20 +

_{0-Fe(IV)-E(.+)}

H202 +

_{0-Fe(IV)-E(.+)}

- H20 +

_Fe(III)-E

+ 02

Here

_Fe()-E

_represents the iron centre of the heme group attached to _{the enzyme.}

Fe(IV)-E(.+)

is a _mesomeric form _of

_Fe(V)-E,

_meaning that iron is _{not completely} oxidized to +V but receives some T'supporting _electron'' A.om the heme ligand. This

As hydrogen _peroxide enters' the active site, it interacts with the amino acids

Asn147

_(asparagine

at position

147)

and His74, causing a _proton

_{(hydrogen ion)}

to transfer between the _oxygen atoms. The free oxygen atom coordinates, freeing the newly-formed water molecule and

Fe(IV)-0. Fe(IV)-0

reacts _with a _second

hydrogen _{peroxide molecule} to _reform

_Fe(III)-E

rand _{produce water and oxygen.} The reactivity of the iron center may be improved by the presence of the phenolate ligand of Tyr357 in the fifth iron ligand, which can _{assist in the oxidation of the}

Fe(III)

to

Fe(IV).

The _{efflCiency of the reaction may also be} improved by the interactions _of His74 _and Asn147 _{with reaction} intermediates. _{In general, the} rate of the reaction can

be determined by the Michaelis-Menten _equation.

Catalase can _{also oxidize} different toxins, _such as formaldehyde, formic _acid,

phenols, and alcohols. In doing so, it uses hydrogen _{peroxide according} to the

following _reaction:

H202+ H2R -> 2H20 + R

Again, the exact mechanism of this reaction isnot known.

Any heavy _metal ion

_(such

as _{copper cations} in

_{copper(II)sulfate)}

_will act as a

noncompetitive inhibitor on _catalase. Also, the _{poison cyanide} is a _competitive

inhibitorl of catalase, strongly binding to the heme of catalase and stopping the enzymeTs action.

Three-dimensional _protein structures of the peroxidated catalase intermediates are

available at the Protein Data Bank. This enzyme is commonly used in laboratories as

a tool for leaming _{the effect of enzymes upon reaction rates.}

Hydrogen _peroxide is a harmful by-product _{of many normal metabolic processes:}

To _prevent damage, it must be quickly converted into other, less dangerous

substances. To this end, catalase is frequently used by cells to _{rapidly catalyze} the

decomposition _{of hydrogen peroxide} into _{less reactive gaseous oxygen and water}

molecules.

The true biological _{signiflCanCe Of catalase} isnot always straightforward to assess:

Mice _{genetically engineered} to lack _catalase are _{phenotypically normal,} indicating

that this enzyme is dispensable _{in animals under} some _conditions.

Some human beings have _very low _{levels of catalase}

_{(acatalasia),}

_{yet show} few ill effects. It is likely that the predominant scavengers of H202 in normal mammalian cells are _{peroxiredoxins rather than catalase.}

Catalase _works at an _optimum temperature _{of 37} oC, _which is approximately _the

temperature _{of the human} body.

Catalase is _usually located in a _{cellular organelle called the peroxisome.}

Peroxisomes _{in plant cells} are involved in photorespiration

(the

use _{of oxygen and}

production of carbon

dioxide)

and symbiotic nitrogen fixation

(the

breaking apart of diatomic _nitrogen

_P2)

tO _{reactive nitrogen}

atoms).

Hydrogen _{peroxide is used} as a _{potent antimicrobial agent when cells} are infected

with a _pathogen. Pathogens that are _{catalase-positive, such} as

_{Mycoba;terium}

tuberculosis, Legionella _{pneumophila, and} Campylobacter

_jejuni,

_{make catalase} in order to deactivate the peroxide radicals, thus allowing them to _{survive unharmed} within the host.

All known

_animals

use _catalase in _{every organ, with particularly} high

concentrations occuming in the liver. One unique use _{of catalase} occurs in

bombardier beetle. The beetle has two sets of chemicals ordinarily stored separately

in its paired glands. The larger of the pail, the storage chamber or _{reservoir, contains}

hydroquinones _and hydrogen _{peroxide, whereas the smaller of the pair, the reaction}

chamber, contains catalases and peroxidases. To activate the

_spray;

the beetle _mixes the contents of the two compartments, causing oxygen to be liberated from hydrogen

peroxide. The oxygen oxidizes the hydroquinones and also acts as _{the propellant.}

Catalase _{is also universal among plants,} but _{not among} fungi, _although some

species have been found to produce the enzyme when growing in an _environment

with a low _{pH and} warm temperatures.

Very few _{aerobic microorganisms} are known that do _not use _catalase.

Streptococcus _species are an _{example of aerobic} bacteria that do not possess catalase.

Catalase has _also been _observed in some _{anaerobic microorganisms, such} as

Methanosarcina barkeri.

Catalase _{is used in the food industry for removing} hydrogen _peroxide &om _milk

prior to cheese production. Another use is in food _{wrappers, where} it prevents food

from _oxidizing. Catalase _{is also used} in the textile industry, _removing hydrogen

peroxide from fabrics to make sure _{the material} is peroxide-free. A _minor use is in

contact lens hygiene - _a few lens-cleaning

products disinfect the lens _using a

hydrogen _{peroxide solution;} a _{solution containing catalase} is then used to decompose

the hydrogen _peroxide before _{the lens is used again. Recently, catalase has also begun} to _{be used in the aesthetics industry.} Several _mask treatments _combine the _enzyme with hydrogen peroxide on the face with the intent of increasing

cellular oxygenation

1.3 Research the response of the

Btjidobactert'um

to Oxygen

In de Vries et al. study, bifldobacteria were divided into three groups

(84).

The fact that none _{of the}

_{Bljidobacterium}

_{strains used} in the present investigation _grew on

agar

_{plates under aerobic conditions,} is in accordance with the.observations ofDehi T. and S. Bald et al.. It is evident that bifldobacteria cannot tolerate the 0- tension _{of air.}

Accumulation _ofH202 Seems tO be a _minor factor in explaining _{the high}

sensitivity of

ttvo other

Bljidobacterium

strains tor02

_(S

822 _and

i

328, _group

_III).

Despite _{of the} fact that small amounts ofH202 Were formed by cell suspensions and cell-free extracts

of these strains, no H202 could be detected in aerated cultures. The partial

inactivation _{of fructose-6-phosphate phosphoketolase observed in aerated cultures}

was _{presumably caused} by traces _{of intracellular} H202. Even _{in the absence of 02,}

concentrated cell suspensions of these strains do not succeed in establishing the oxidation-reduction potential required for fermentation of glucose. Addition of

cystcine or _{ascorbic acid lowers} the oxidationreduction

potehtial

to the required value. This is true for both _{anaerobically-grown cells} as _{cells from aerated cultures. In the}

absence of cysteine, anaerobic growth of these strains is slow. Cells do not succeed in

bringing _{the oxidation-reduction potential of the medium} to a _{value suitable} for more

rapid growth until the concentration of the cells exceeds a _{given value.} The1

oxidation-reduction potential obtained finally in such a _culture is very low. From the

results, it is evident that does not exert a lethal action on these _strains

(group III),

but

prevents growth by establishing a too high _{oxidation-reduction potential.}

₍₁

8, 38, 46,

47,77,89)

In Kawasaki S. et al. study, B. boum and B. thermophilum _{show growth}

conditions, so they are _{reasonably classified} as _anaerobes. The _{accumulation of H202}

in 02-Sensitive _species must be the end product of 02 reduction. NADH-dependent

oxidase activities were detected as _{part of} an 02 _{reduction system.} Although the total

activity of NADH dependent oxidase in the CFE was _{similar among species,} the

activity profiles differed between 02-Sensitive and microaerophilic species. The

growth inhibition ofB.

bljidum

and B. longum under 20% 02 conditions was _partially

reversed when catalase was added to the medium. The inability of exogenously added catalase to decompose intracellular H202 might be a reason for the

_failure

to _obtain

complete growth recoveries. These results indicate that the primary factor in aerobic inhibition _{is the production ofH202} derived from _{02 reduction.}

_(39,40)

Oxidative _stress can be defined as an excess _{of reactive oxygen species}

(ROS)

that have _{strong oxidizing potential} for tells. ROS cause damage to _{macromolecular}

constituents such as DNA, RNA, _{proteins and} lipids. Toxicity occurs _when the degree of oxidative stress exceeds the capacity of cell defence systems. ROS originate &om partial.reduction of molecular oxygen

(02)

tO SuPerOXide

(02-),

hydrogen peroxide

(H202)

and hydroxyl radical

(OH. ).

The biological sources _ofROS are numerous, _e.g.

they can be _generated in aerobiosis by flavoproteins, _and by _macrophages during

inflammatory _reactions. Thus, _oxidative stress plays an important _{role in pathologies}

of the gastrointestinal tract ofhumans such as inflammatory bowel diseases

and in the

radio-induced tissue

injury

that may occur during _{radiotherapy.}

As _probiotics, some _{species of}

_{Bljidobacterium}

are _{widely used} in _various

therapeutics _and food _products. However, as bifidobacteria are _{obligate anaerobic}

bacteria, _{their sensitivity} to 02 _{limits their manufacturing and storage.} Bifidobacteria have an _oxidase function that uses 02 aS an _{electron acceptor} to _reduce to H20 _and

aerobic growth inhibition in bifidobacteria. Although NADH peroxidase was found in

bifldobacteria _and some types _{of peroxidases} were _predicted, including thiol

peroxidase, alkyl hydroperoxide reductase, and peptide methionine sulfoxide

redhctase,

these peroxidases are _unable to decompose the H202 _Produced by

bifldobacteria _{under aerobic conditions.} In _addition, we _{could not} detect H202

scavenging activity in B. longum 105-A.

Superoxide dismutase, _{catalase, and glutathione peroxidase}

_(GPX)

_{constitute the} enzymatic antioxidant system. GPX and catalase are _{the enzymes} that can decompose

H202 tO H20. GPX was _{mainly studied} in eukaryotic _organisms; however, little is

known _about GPX _{activity in prokaryotic organisms, and} a GPX _gene has _not been

detected _{in the genomic sequences ofbifidobacteria. In addition, bifidobacteria} do not

carry glutathione synthesize pathway. Catalase is an _{enzyme commonly} found _in

aerobic bacteria but is absent in almost all anaerobic bacteria

lincluding

Bljidobacterium.

B. subtilis KatE

_{(heme-dependent catalase)}

isa _{well-known catalase}

that is used to improve _{the viability of} some _{species of bacteria via heterologous}

expression

(13,

50,

63).

In this study, we investigated the effects of expressing B. subtilis heme-dependent catalase on the

_oxidative

stress resistance of B. longum

1 05-A. For _comparison, the effects ofAexogenously _added cafalase on B. longum were

Materials _and Methods

2.1 Medium _and Buffer

E. coli and B. subtilis were _grown in LB _{medium at} 37oC. For _{anaerobic culture, B.}

longum 105-A was _grown in MRS _medium

(Difco,

Franklin, New

_Jersey)

_containing

0.34% L-ascOrbic acid sodium salt

Papalai

Tesque, Kyoto,

Japan),

0.02% _L-cysteine

pacalai Tesque),

and 50 mM sucrose at 37oC. For aerobic culture,I B. longum 105-A

was _grown in MRS _{medium containing} 50 _mM sucrose _and 10 _LIM hemin

(Sigma-Aldrich,

St. Louis,

_MO)

at 37oC. When necessary, antibiotics were _{used at} the following _{concentrations: chloramphenico1}

_(Cm;

25

LLg/mL),

kanamycin

(h;

50

pg/mL)

and spectinomycin

(Sp;

75

_LLg/mL).

Aerated cultures

(10 mL)

of

Bljidobacterium

were _grown in 25-mL _{tubes using silicone plugs}

with shaking at 120

rpm. Anaerobic cultures

(10 mL)

were _grown in 12-mL test tubes _with screw _caps.

CFUs _ofB. longum 105-A were _counted as follows: _{appropriate sample} dilutions were

prepared in sucrose _solution

(50

_mM sucrose _{and 1 mM} triammonium _{citrate, pH}

_6.0),

plated on MRS _{agar, and} incubated for 48 h _at 37oC

under anaerobic conditions using

the AnaeroPack system

(Mitsubishi.

Gas Chemical, Tokyo,

Japan).

2.1.1

_{LuriaBrothmedia(LB)}

Trypto ne

Yeast extract

Agrose

2.1.2 MRSmedium

Difco Lactobacilli MRS Broth

...0.5%

*Approximat6 Fomul Per Liter Protesose Peptone Beef Extract Yeast Extract Dextrose Polysorbate 80 Ammonium Citrate Sodium Acetate Magnesium _sulfate Manganese _sulfate Dipotassium _phosphate

L-Ascorbic Acid Sodium Salt

_(Vc)

L- Cysteine Agrose 2.1.3 TEbuffer

Tris-HC1(pH8)

EDTA(pH

8

₎

2.1.4 PEG Polyethylene _glycol 6000 *

Use a brown bottle in one _month.

2.1.5 PBSbuffer KC1 0.34% 0.02% 1.5%

_{(ifnecessary)}

1 0mM 0.02%

Na2HPO4

KH2PO4

0.144%

0.024%

*Adjust

the pH to _{7.4 with HC1.}

Because _{that the solution} can't be stored, so _{the concentration} be 10 x to _stored.

2.2 Isolate DNA from _{microorganisms}

Using UltraClean Microbial DNA Isolation kit.

2.3 Extractplasmid

2.3.1 With

_QIA

_{prep spin Mini prep} kit Using

_QIA

_{prep spin Mini prep} kit.

2.3.2 With 2-propano1 2.3.2.1 Thereagent 2.3.2.1.1 SolutionI

Tris-HC1(pH8)

EDTA(pH8)

50mM 25mM 1 0mM

*Confect the lM _ofTris-HCl to store.

Ajust

pH with lM ofNaOH. Confect the lM _ofEDTA to store.

_Ajust

pH with lM ofNaOH.

NaOH

*Because that the solution can't be stored, so _{the concentration} be 10 x to

stored. When useing it, provisional confect. SDS is the last one _added into

the tube.

SDS _:sodium dodecyl _sulfate

2.3.2.1.2 SolutionIII Potassium

_Acetate(5M)

Acetate 60mL ll.5mL 28.5mL 2.3.2.2 Protocol

1. Transfer _{the microbial culture} to a _{microcentrifuge} tube.

2. Centrifuge for 2 min at 12,000 rpm

(-12,000

x

g),

discard the supematant. 3. Add 200 _{pl Solution I and mix thoroughly by vortexing.}

4. Add 400 _{Lil Solution II and mix thoroughly} by inverting the tube 4-6 times.

5. Add 300 _{pl Solution III and mix thoroughly} by inverting _{the tube 4-6 times.}

6. Wait _{5 min.}

7.

_C)entrifuge

_{for 10 min} at 12,000 _rpm

_(-12,000

x

g),

transfer the supernatant to

a new _{microcentrifuge tube.}

8. Add _{lLtl of 10 mg/mL} RNAse _{into the microcentrifuge tube.}

9. Incubate _at 37oC for 30-60 _min.

times.

ll. Centrifuge _{for 15 min} at 12,000 rpm

(-12,000

x

g),

transfer the supernatant

to a new _{microcentrifuge} tube.

12. Add 500 _{lil of} 70% _{ethanol and mix} thoroughly by inverting _{the tube} 2

times.

13. Centrifuge for 10 min at 1'2,000 rpm

(-12,000

x

g),

discard the supernatant. 14. Centrifuge for an _additional 1 min at 12,000 rpm

(-12,000

x

g)

to remove

residual ethano1.

15. Dry _{the pellet in the incubator} or in the aspirator.

16. About _{15 min} later, dissolved _{in 50 lilofTE} buffer. Store p.1asmid fi.ozen

_(-20oC).

2.4 PurifyDNA

2.4.1 With Nucleo Spin Extract II kit

using PCR _clean-up Gel _extraction NucleoSpin@ Extract II. MACHEREY-NAGEL

kit.

2.4.2 With Ethano1

2.4.2.1 Thereagent

lI3M Sodium Acetate buffer, pH 5.2

(stole

at 4

oC)

lICold 100% Ethano1

(-20oC)

tCold 70% Ethanol in sterile dH20

(-20oC)

)DNA sample

4 oC Microc'entrifuge

_(normal

_{microcentrifuge in cold} room _works

fine).

All

centrifugations should be on "soft''

(no brake)

_setting.

2.4.2.2 Protocol

1. Transfer DNA to a _{container where} it f111sone fourth _{the total volume.}

(a

₅₀₀

pl tube should have no more than 125 _{Lil ofDNA solution,} for

_example)

2.I Add one tenth _volume of Sodium Acetate buffer to _equalize ion

concentrations.

3. Add at least two _{volumes ofcold} 100% _ethano1; let stand in

-20oC freezer for

at least one hour.

4. Centrifuge _{sample for 15 minutes} at highest speed in a 4oC _{microcentrifuge.}

5. Remove as _{much supernatant} as _{possible with} a 1 mL _{micropipet; recentrifuge,}

then remove the rest with a ₂₀₀

pl pipet.

6. Add 200 _{pl ofcold} 70% _{ethano1; centrifuge for 5 minutes} in a 4 oC centrifuge.

7. Remove _{supernatant with} a 200 _{Lil pipet; evaporate remaining ethanol} in a

37 oC water bath.

8. Resuspend _{pellet in desired volume of} water or TE buffer.

2.4.3 With PEG

2.4.3.1 The _reagent

LPEG

A70% Ethanol in sterile dH20

(-20oC)

IIDNA sample

1. Mix _{1 volume ofDNA sample with 1 volumes of PEG.}

2. Incubate _at 4oC _{for 30 min.}

3. Centrifuge _{for 15 min} at4oC _with 15,000 _rpm, discard _{the supematant.}

4. Add 500 _{Lil 70% ethanol and} don't _mix.

5. Centrifuge _{for 15 min at}4oC _with 15,000 _rpm, discard _{the supematant.}

6. Centrifuge for 1 min at4oC with L15,000 rpm to remove the residual ethano1.

7. Dry _{the pellet in the incubator} or in the aspirator.

8. About _{1 5 min later, resuspend pellet in desired volume ofddH20.}

2.5 Preparation _{of competent cell}

2.5.1 Electrocompetent _{cell ofBifidobacterium}

2.5.1.1 Thereagent

Sucrose buffer

Sucrose

1 00mM Ammonium _citrate

_bH6.0)

*

Ajust

pH

&ith

lM citrate. Autoclave 121oC _{for 20 min.} MRS _medium

1.712%

2.5.1.2 Protocol

1. Incubate the Bifidobacteria at 37oC with anaerobic culture by MRS medium.

3. Transfer _{10 mL culture} to the tube.

4. Centrifuge for 10min _at4oC _with 6,000 _rpm, discard _{the supernatant.}

5. Add _{10 mL} sucrose buffer _{and mix} thoroughly by vortexing.

6. Centrifuge for 10min _at4oC _with 6,000 _rpm, discard _{the supernatant.}

7. Add _{5 mL} sucrose buffer _{and mix} thoroughly by vortexing.

8. Centrifuge for 10min at 4oC with 6,000 rpm, discard the supernatant

remaining 1 mL.

9. Transfer _{50 lil}to a new _{microcentrifuge.}

10. Transfer the tube to ice. Use it in 1 hour.

2.5.2 Chemically _{competent cell ofE.coli}

2.5.2.1 Thereagent

0.1 M CaC12

LB _medium

2.5.2.2 Protocol

1. Incubate _{E. coli} at37oC with 120 rpm shaking.

2. When OD660-0.5, _{stop culture.}

3. Transfer _{1.5 ml culture} to a _{microcentrifuge tube, and chill} on ice for ₁₀

minutes.

4. Pellet the cells by centrifugation at 3000 x _{g for 5 minutes at} 4oC, discard _the

Sup ernatant.

6. Incubate tubes on ice for 20 minutes.

7. Pellet the c-ells

-as

before(3000

x _{g, 5 minutes,}

_4oC),

discard _{the supernatant.}

8. Add 100 _{pl cold 0.1 M} CaC12 _{and mix} throughly _{by mild vortexing.}

Store _{the competent cells frot2en}

_(-80oC).

2.6 SDS-PAGE

2.6.1 Thereagent

Acrylamide / bisacrylamide monomer _{stock solution}

(30%

/

0.8%)

Acrylamide monomer

N,N-methylenebi _sacrylamide

4 x Running _{gel buffer}

(1.5

M Tris-HCl,

pH8.8)

Tris-HC1

*Ajust

pH with HC1. Store at4oC.

4 x Stacking _{gel buffer}

(500

_mM Tris-HC1,

pH6.8)

Tris-HC1

*Ajust

pH with HC1. Store at4oC.

5 x Sample _Buffer Glycerol Tris-HCl

_bH6.8)

Bromophenolblue 29.2% 18.15% 0.2mM 0.05%

SDS _{stock solution}

_(10%)

APS-Ammonim _{persulfate initiator solution}

_(10%)

Ammonium _{persulfate...} TEMED-N, N, NT, NT-tetramethylethylenediamine 1 x Running buffer Tris-HCl Glycine CBB _{stain solution} CBB-R250 Methanol Acetic _acid 25mM 200mM 0.25%

Protein MW Maker

_{(Daiichi) from}

top to bottom

1. 97,400; 2. 66,267; 3. 42,400; 4. 30,000; 5. 20,100; 6. 14,400

CBB destain _solution

Acetic _acid

Methanol

2.6.2 Protocol

1x Running Gel Solution H20 1.5 M Tris-HCl, pH 8.8 10% _(w/v)SDS AcrylamideBis-acrylamide (30%/0.8% _w/v) 7 0/o 10 0/o 15.3 ml 12.3 ml 7.5 ml 7.5 ml 0.3 m1 0.3 ml 6.9 ml 9.9 ml 120/o 150/o 10.2 m1 7.2 m1 7.5 ml 7.5 m1 0.3 ml 0.3 ml 12.0 ml 15.0 ml

10% _{(w/v)ammonium persulfate (APS) 0.15 m1} 0.15 m1 0.15 ml 0.15 ml

TEMED 0.02 m1 0.02 ml 0.02 m1 0.02 m1

Stacking Gel Solution

_{(4% Acrylamide)}

II20

0.5 M Tris-HCl, _pH 6.8

20% _(w/v)SDS

Acrylamidemis-acrylamide (30%/0.8% w/v)

10% _{(w/v)ammonium persulfate (APS)}

TEMED 3.075 ml 1.25 ml 0.025 _m1 0.67 m1 0.025 _m1 0.005 _ml

Choose a _{percentage acrylamide} based on _{the molecular weight range of proteins}

you wish to separate:

% Gel M.W. Range

50 kDa- 500kDa

12 10 kDa- 200 kDa

15 3kDa- 100kDa

2. Preparing Sample

Mix _protein :5 x _samplebuffer :2-mercaptoethano1 - 20

:5 :1.

Heat _sample by boiling for 5-10 _minutes.

Cool down the temperature on ice.

Centrifuge _{the sample and} use the supernatant.

3. Loading _samples on _gel

Pull the combs out- straight upwards.

Wash-out _{the wells and fill the wells with 1} x _Running buffer.

Add _{the samples and protein} MW _{marker into the wells}

4. Electrophorese

When _{the samples in the stacking gel,}

adjust

20 mA for -30 min.

The _samples into the running gel,

_adjust

to 30 mA until the samples move to the bottom _{of the gel}

5. Finished Gel: Remove from Rig

6. Staining _{the gel with CBB stain solution for 30 min.}

7. Destaining

Add CBB destain _{solution and shaking ovemight.}

Remove de-staining _{solution, rinse gels twice with dH20.}

8. Take the photo.

2.7 Detection _{activity of catalase}

2.7.1 Add H202 tO detect _{activity ofcatalase}

2.7.1.1 Thetheory

Catalase

_(EC

1.1

_1.1.6),

_{present in the peroxisomes of nearly all aerobic cells,}

serves to protect the cell from the toxic effects of hydrogen peroxide

by

catalyzing its decomposition into molecular oxygen and water without the

production of fi:ee radicals. The mechanism of catalysis is not fully elucidated, but the overall reaction is as

follows:

2H202--2H20+02

The _{presence of catalase activity leads} to bubble formation _resulting from the

transformation ofH202 tO H20 and gaseous 02.

2.7.1.2 Protocol

1. Incubate the bacteria until-OD660 _-1.0.

2. Transfer _{1 5 mL of the bacteria culture} to a _{microcentrifuge tube.}

3. Centrifuge _{for 10 min} at 12,000 g and discard the supernatant.

4. Add lmL PBS buffer _{and mix} thoroughly _{by vortexing.}

6. Resuspend _{the pellet with 30 pl PBS} buffer.

7.

_{Samples(20-pl)}

_{ofPBS resuspended cells} were _{mixed with} 10 ml

_{H202(8.8 M)}

2.7.2 Assayofcatalase 2.7.2.1 Thereagent Dichromate _solution Potassium dichromate Acetic _acid PBS buffer H202 0n _{Various concentrations} 1.67% 6.67% 0.1mM _-1mM 2.7.2.2 Protocol

Draw _{the standard} curve

1. Transfer 900 _{pl dichromate solution} to a _{microcentrifuge} tube.

2. Add 300 _{pl H202 0fvarious concentrations and mix} thoroughly _{by vortexing.}

3. Heat _sample by boiling for 5-10 _minutes.

4. Down the temperature to -25oC.

5. Measure _{the sample} at OD570.

6. Drawing _{the standard} curve.

Assay _{of the samples}

1. Transfer 900 _{Lil dichromate solution} to a _{microcentrifuge tube.}

3. Bacteia _samples

_(109c.f.u.)

are _{mixed with} 0.88 mM H202 in PBS buffer.

4. Transfer 300 _{lil of the mixture} to the tube _{and mix} thoroughly by vortexing.

5. Heat _sample by boiling for 5-10

minLtes.

6. Down the temperature to _-25oC.

7. Measure the sample at OD570.

8. Calculate the data base on the standard curve.

2.8 Short-term H202 _exposure 2.8.1 Thereagent MRS _medium PBS buffer Hemin H202 C atalase Sucrose buffer Sucrose 1 00mM Ammonium _citrate

_bH6.0)

*

Ajust

pH with lM citrate. Autoclave 121oC _{for 20 min.}

...10U

1.712%

2.8.2 Protocol

1. Incubate the Bifldobacterium with MRS + _{10 LIM hemin.}

2. Transfer _{1 mL culture} to a _{microcentrifuge} tube.

4. Add _{1 mL} PBS buffer _{and mix} thoroughly by vortexing.

5. Centrifuge _{for 10 min} at 12,000 x _{g and} discard the supernatant.

6. Add _{1 mL} MRS _{medium and mix} thoroughly by _vortexing.

7. Incubate at 37oC with 4.4 mM H202 for 1 h. *Negative: With 0 M H202.

8. Remove the H202 by addition ofcatalase

(10

U

mL-1).

9. Spread desired dilution _{volume of culture} on MRS _{plate and} incubate for 48

hours.

10. Take count of the colonies.

2.9 Long-term with_aerated _cultures

2.9.1 Thereagent

MRS _medium

Hemin

2.9.2 Protocol

1. Anaerobic incubate the Bifidobacterium _with MRS.

2. Inoculate 160 _{pl Bifidobacterium} to 8 mL MRS not contain Vc with aerobic method.

3. Incubate at 120 rpm and spread desired dilution volume on MRS _plate at 12-h

intervals during 2 days.

2.10 PCR

2.10.1 With _po1ymerase KOD-PLUS

Using KOD

-Plus- kit.

2.10.2 With _po1ymerase GOTaq

using GoTaq@ DNA kit.

2.ll DNA _extraction from _{agarose gels}

using PCR _clean-up Gel _extraction NucleoSpin@ Extract II. MACHEREY-NAGEL

kit.

2.12 Digestion with _{restriction enzyme}

2.12.1 The _{restriction enzyme}

The _{restriction enzymes} are _applied by TAKARA or NEB.

2.12.2 Protocol

Combine the following in a _{microfuge tube in order} :

1LL1 10 x Buffer

6.5 lilH20 2 lilDNA 0.5 LilEnzym9

2.13 DNAligation

Using DNA Ligation kit < Mighty Mix > kit.

2.14 Transformation 2.14.1 Chemical transformation 2.14.1.1 Thereagent Competent _cells Plasmid S.0.C _medium or LB _medium Selective _plate Ampicillin

_(AmpR)

Kanamycin

_(KmR)

spectinomycin

_(SpR)

chloramphenico1

_(CmR)

1 00pg/mL 50pg/mL 75 pg/mL 25 pg/mL 2.14.1.2 Protocol

1. Thaw, on ice, one _{vial of chemically competent cells for each} transformation.

2. Add _{1 lilplasmid}

_(-50ng)

into a _{vial of chemically competent cells and mix}

gently. Do not mix by pipetting up and down. 3. Incubate the

_vial(s)

on ice for 30 minutes.

4. Heat-shock _{the cells for 30 seconds} at 42oC _without shaking.

6. Add 250 _{pl of} room temperature S.0.C. _medium

_(or

LB

_medium)

to _{each vial.} 7. Cap the

_vial(s)

tightly and shake horizontally

(120 rpm)

at37oC for 1 hour. 8. Spread 20 _{pl and} 100 _pl

kom

_each transformation on a _{prewarmed selective}

plate and incubate ovemight at 37oC. We recommend plating 2 different

volumes to ensure that at least 1 plate has well-spaced colonies.

2.14.2 Electroporation 2.14.2.1 The _reagent MRS _medium Competent _cells Plasmid Selective _plate * Th'e same as 2.14.1.1. 2.14.2.2 Protocol

1. Add _{1 pl plasmid}

_(-50ng)

into a _{vial of competent cells}

(-50p1)

_{and mix}

gently. Do not mix by pipetting up and down. 2. Incubate the

_vial(s)

on ice for 15 minutes.

3. Prepare the tube _added 1 mL of MRS medium.

4. Transfer the mixed competent cells to a 2-mm cuvette.

5. Electroporation was _{camied out with}

EasyjecT (EQUIBIO),

_{set at} 12kV/cm

(time

constants obtained between 3.9 and 4.2

_ms).

6. Transfer _{the cells} to the tube _added MRS _medium. 7. Incubater the tube ar 37oC for 3 hours.

incubate at 37oC for 2-3 days. We recommend plating 2 different volumes to

ensure that at least 1 plate has well-spaced colonies.

2.15 Gateway _system

using MultiSite Gateway@ Pro kit.

2.16 Flow _cytometry

2.16.1 Thereagent

Using LIVE/DEAD@ BacLightTM Bacterial Viability _and Counting kit.

2.16.2 Theinstrument

cell Lab

_QuantaTM

SC MPL.

_Betkman

c.ulter.

2.17 Isolate NA from _{microorganisms}

Using RNeasy Mini kit.

2.18 RT-PCR

Using TURBO DNA-free kit; High Capacity _CDNA Reverse Transcription kit.

2.19 _qRT-PCR

Using SYBR Premix Ex Taq kit.

2.19.2 The instrument

StepOnePlus Real-Time PCR _system. Applied Biosystems.

2.20 AssayofH202 2.20.1 Thereagent Triton X-1 00 Horseradish _peroxidase o-dianisidine dihydrochloride PBS buffer 2.20.2 Protocol 0.01% 0.63mM

Culture _supernatants were diluted 3:2 with a _{solution of 0.2%} Triton X-100, 0.01%

horseradish _{peroxidase, and} 0.63 _{mM o-dianisidine} dihydrochloride _{in 50 mM} acetate

buffer, _pH 5.0. The _{accumulation of} H202 in the _{culture medium} was _assayed

spectrophotometrically at 460 nm by detecting _{the oxidation of o-dianisidine}

dihydro _chloride.

using KOI) plus Mutagenesis kit.

_(Process

see

below)

prCA:mLASE )

Patabsq&--1

atalas

tfLD5

i

Dpn.

I.P7c:T:01

i_

pB CATOO1

_/

L-LL Ligation

//I

Tl

pBCATOO1 ::FtBSn Catala

/

･-GCTT

RBSn

1

ATGAG...

Cata[ase

2.22 The _{strains, the plasmids and primers}

Strains, plasmids or _primers Characteristic(s)

Bacterial _strains B. longum 105-A

Bacillus _{subtilis GTCO} 1672 E. coli DH5a

E. coli One Shot MachlTM TIR E. coli One Shot ccdB SurvivalTM TIR E. colt UM255 Plasmids pDONRTM22 1-P 1-P5r pDONRTM22 1_-P5-P2 pDOm1 5r: :HpkatE pDONR52: :hupT pKKT427 pGU100 Primers kat- f kat-r hup-f hup-r attB 5 -hupTf attB 2-hupTr prRB Sh9 prRBSh8 prRB Sh7 prRB Sh6 prRB Sh5 prRB Sh4 prRB Sh3 prRB Sh2 prWS1 1 prRBS 10 Wild type Wild type

F- endAl glnV44 thi-1 recAl relAl gy7A96 deoR nupG p80dlacZ4M15

A _PacZYA-argF) U1 69, hsdR1 _7jrK-mK'),

L-

F-p80PacZ)AM15 AlacX74 hsdR(rKmK+) ArecA1398 endAl tonA

F- mcrA A(mrr-hsdRMS-mcrBC) 80lacZAM15 AlacX74 _{recAl araA139}

D(ara-leu)7697 _{galUgalK rpsL} (StrR)endAl nupG tonAI:Ptrc _-ccdA

pro leu rpsL hsdM hsdR endl lacy kalG2 katEI:Tn10 recA

pDONRTM221; CmrKmr; ccdB; attP1; attP5r pDONRTM22 1;Cmr Kmr; ccdB; attP5; attP2

PDO-TM221-P 1-P5r; Kmr; carrying theB. subtilis katE ;hup promoter PDO-TM221-P5-P2; Kmr; ca-rrying the hup terminator

Spr; A _{shuttle vector} between E. _{coli and Bljidobacterium,}3.9 kb modiflCation of pBRATA1 0 1

pKKT427; Cmr, Spr; ccdB; Reading Frame Cassette A

atgagtgatgaccaaaaca

ggggaca acttttgta tacaa agttgttc aaattc gtc tatcc caat

I

gggga caagtttgta ca aa aaagcaggcttttc c_gccactttg ct ttggtcatcactcataaaagcatccttcttggg

gggga ca actttgta taca aa agttgc cttctgctc_gtagcg atta gggga cca ctttgta ca aga a_agctgggta tggaag c_gctgaac tagtc c

AATAAAGCATCCTTCTTGG AAAAAGCATCCTTCTTGGG AAJuG CATCCTTCTTGGGT AAAGCATCCTTCTTGGGTC AAGCATCCTTCTTGGGTCA AG CATCCTTCTTGGGTCAG GCATCCTTCTTGGGTCAGG CATCCTTCTTGGGTCAGG G AAGCCTCCTTGGGTCAGGGGACAAGCACTT AAGCACTCCTTGGGTCAGGGGACAAGCACTT

prRBS9 prRBS8 prRB S 7 prRBS6 prRBS5 prRB S4 prRB S3 prRB S2 prRBS 1 prCATALASE juGCATCTCCTGGGTCAGGGGACAAGCACTT AAGCATTCCTTGGGTCAGGGGACAAGCACTT AAGCATCTCCTTGGGTCAGGGGACAAGCACTT AAGCATCTCCTTTGGGTCAGGGGACjuGCACTT AAGCATCCTCCTTTGGGTCAGGGGACAAGCACTT AAGCATACCTCCTTTGGGTCAGGGGACAAGCACTT AAGCATCACCTCCTTTGGGTCAGGGGACAAGCACTT AAGCATACCTCCTTTCGGGTCAGGGGACAAGCACTT juGCATACCTCCTTTCTGGGTCAGGGGACAAGCACTT ATGAGTGATGACCAAAACAAACGTGTAAATGAACACTCAA a

cmr ,Kmr and Spr, resistance to chloramphenicol, kanamycin, and spectinomycin, respectively. b

Results

3.1 Isolate DNA froJn miCrOOrgamiSmS

Isolate DNA &om microorganisms:

strain No. Biosafety level

Bacillus _subtilis GTCO1672 BLSI

Bljidobacterium

longum 1 05-A BLS 1

Bacillus _subtilis was _grown in LB at 37oC.

Bljidobacterium

longum 1 05-A was _grown in MRS at 37oC.

Isolated DNA _{of Bacillus subtilis}

(0.8%

agarose, 1 00v, 30min, Fig. 1

a).