iscussion

under anaerobic conditions

(OD660

^‑

1.0),

whereas the extracellular catalase activity less than 0.4%.

(53,

^{56, 57,}

85)

We investigated the effect of KatE ^on the short‑term H202 tolerance of B. longum

105‑A. The survival ^rates ofB. longum 105‑A

bBCATOO1, pKKT427)

^{were detehmined}

by incubating cultures for 1 h in MRS medium with 4.4 mM H202 _at 37oC. The survival

/

rates of B. longum 105‑A

bBCATOO1)

^during the exponential and stationary phases

were significantly increased by 120‑ and 103‑fold, respectively, compared ^to that ofB.

longum 105‑A

(pKKT427).

^{The data in Table} 1 illustrate that exponential‑phase cells

were more sensitive ^to H202 than stationary‑phase cells.

(50)

We also investigated the physiology ofB. longum 1 05‑A under aerobic conditions. B.

longum 105‑A

bKKT427)

reached ^a maximum growth ^{rate at} 12 h aRer inoculation under anaerobic conditions. The growth ofB. longum 1 05‑A

(pBCATOO1)

^was partially inhibited and B. longum 105‑A

bRT427)

^was nearly stopped under aerobic culture conditions

(Fig. 14).

^{To measure growth rates, cells were cultured and then}

plate‑counted. Although most of the B. longum 1 05‑A

bKKT427)

specimens survived 12 h of aerobic culture, cell growth began to sharply decrease and became almost

unculturable aRer 24 h in aerobic culture. However, B. longum 105‑A

bBCATOO1)

exhibited high survival

(1x107

colony‑forming units

(CFU)/mL)

^{at 24 h} and only became unculturable after 48 h in aerobic conditions

(Fig.15).

^The presence of KatE protected B. longum 1 05‑A from aerobic culture‑induced death.

The concomitant generation of H202 ^Was also measured

(Fig.17).

^The accumulation of H202 in B. longum 105‑A

(pKKT427)

^{increased for} 18 h and peaked at 0.1 mM.

H202 Was scavenged by the genetically expressed catalase during the exponential phase,

and it did _not begin to accumulate in the medium ofB. longum 105‑A

bBCATO'01)

until the stationary phase. At this time, the cells became unculturable

(Fig.

¹

5).

Interestingly, the decrease in the growth of B. longum 105‑A

bKKT427)

^was faster than that of B.

longum 105‑A

bBCATOO1). This

might be because the concentration of H202 in B.

longum 105‑A

bBRT427)

^{was 2‑fold higher than} that ofB. longum 105‑A

bBCATOO1)

in aerated cultures. B. longum 105‑A

bBCATOO1)

survived longer due to the increased period of time in which H202 had _not accumulated. This difference in growth suggests that H202 ^Was Primarily responsible for B. longum 105‑A becoming unculturable under

aerobic conditions.

Lahtinen _et al. reported that B. longum lost culturability quickly during storage, but

the cells still maintained intact membranes

(47).

^{H202 is known to damage DNA} and protein; however, it is unknown whether H202 Can easily damage the B. longum

membrane. Therefore, we investigated whether it was possible that B. longum 105‑A lost their culturability but maintained ^an intact membrane. These experiments ^were

conducted using the LIVE/DEAD

BacLight

^bacterial

viability

^kit

(L/D; hviLfr.gen).

After 24 h in aerobic culture, B. longum 105‑A containing pKKT427 remained

relatively stable, and lx107 'viable' cells/mL ^were maintained

(Fig. 16);

^{however, the}

survival decreased to lx101‑2

cFu/mL (Fig.15).

^{Based on} this information, ^we were

only able ^to make the decision that the cells had intact membranes, but it is still

unknown whether the cells ^were dead. To conf1rm Whether cells maintain viability, further studies ^are needed, such ^as examining the synthesis ofDNA, RNA, and protein.

Some studies reported that adding exogenous catalase ^to the liquid medium improved

aerobic growth of bifidobacteria. Because H202 readily difhses across cell membranes but exogenously added catalase ^cannot penetrate cell membranes, ^we therefore

compared the culturable B. longum 105‑A protected by catalase expression with the culturable B. longum 1 05‑A protected by the addition of exogenous catalase. Although

the counts of B. longum 105‑A

bKKT427)

recovered when cultured under aerobic

conditions with exogenously added catalase from bovine liver

(100

and 3000 U/mL medium, C1345‑1G,

SIGMA),

^{the counts} ofB. longum 105‑A

bKKT427)

^were similar

regardless of concentration of added catalase. Interestingly, the counts of B. longum 105‑A protected by addition of exogenous catalase ^were nearly identical those of ^to B.

longum

105‑A ^bBCATO61)

^when ae,.bically cultured f.r 18 h, alth.ugh the concentrations of exogenously added catalase ^were much higher than the levels of expressed catalase. B. longum 105‑A that ^was protected by exogenously added catalase

was unculturable aRer 36 h in aerated culture; however, B. longum 105‑A

bBCATOO1)

did _not become unculturable until 48 h in aerated culture. These results indicate that B.

longum maintained intact cell membranes whereas induced exogenously added catalase

eliminated extracellular H202 but was unable ^to eliminate intracellular H202.

H202 Was ^a Primary factor in

Bljidobacterium

^becoming unculturable under aerobic conditions; the addition of exogenous catalase ^or the genetic expression of catalase

could

^protect

Bljidobacterium

^from oxidative ^stress. This effect ^was weaker for exogenous catalase than for heterologously expressed catalase. Further studies ^are

needed ^to find a promoter that can induce higher and prolonged expression of catalase that will allow for complete scavenging of H202 and improve viability under aerobic

conditions.

4.2 Improvement of catalase expression level

Although expression of catalase could protect

Bljidobacterium

^from oxidative ^stress, the expression level was still low. To improve the expression level, we tried ^to improve the transcription and translation of catalase level.

4.2. 1 Transcription

It is difrlCult to obtain high levels of expression of foreign genes in

Bljidobacterium,

some shuttle ^vectors have been reported. The promoters of the gap, a‑galactosidase, lacl family transcriptional regulator, 1 6s rRNA, and hup genes from

Bljidobacterium

^have

been used ^to express foreign genes in B. longum and B. breve. In this study, the B.

subtilis catalase gene could ^not be expressed under the control of its native promoter. To obtain high catalase activity in

Bljidobacterium,

^a strong, organism‑specific promoter

was needed. The gap and ^{hup genes have high codon adaptation indices}

(CAIs)

^{in B.}

longum, 0.742 and 0.629, respectively, and both are highly expressed in

Bljidobacterium.

Homology searches ^were performed for the upstream regions of gap and hup in B.

adolescentis ATCC15703, B. animalis lactis ADOll, B.

bljidum

^{PRL2010, B. dentium}

Bd1, and B. longumNCC2705

(Fig.18).

^The gap and hup upstream regions have a

highly conserved putative ribosome binding site

(RBS).

^By comparison ^to the typical

‑35 and ‑10 promoter ^consensus sequences of TTGACA and TATAAT, TTGGCN and TANTAT were possible candidate for the hup gene ‑35 and ‑10 sequences, respectively.

For the gap gene, the promoter ‑10 sequence, TACAGT, ^was conserved in all strains.

H.wever, TTGCCC

was

^{the putative p,.m.te, ‑35 sequence in all}

Pljidobacte,ium

strains except B. animalis lactis ADOll, whose putative prorpoter ‑35 sequence ^was TTGAAG. In addition, the distance between the putative ‑35 with ‑10 regions of the hup promoters in five

Bljidobacteria

strains and the gap promoter in B. animalis lactis

were shorter than the typical distance; however, the distance between them in the gap promoters in B. adolescentis, B.

bljidum,

B. dentium, and B. longum ^was 1 8 nucleotides.

Since little is known about the po1ymerase in

Bljidobacteria

compared ^to those of other bacteria, further experiments will be required ^to understand the upstream regions of

these genes

(28,

29, 30, 33, 48, 52, 59, 75, 80,

81).

The results of quantitative RT‑PCR for catalase transcript in B. longum 105‑A

bBCATOOl

and

pBCATOO2)

^{are shown on} flg. 19. The quantity of catalase transcript

was close ^to each other under the control of hup and gap promoter either ^at the

exponential phase ^or stationary phase, and the catalase activityalso ^was close

(1.

^{1 8‑fold,}

hup promoter ^vs. gap promoter, Fig.

13).

A

a.dqllium Bdl a.adolescenLis A 7CC 75703 8Jorgum NCC2705

LLbiGdum PRL20 10 a.admalis lacEls JWO1 ¹

CqlSOnSUS

a̲dLZnILum Bdl a̲i)doltrsconLLs A71CC15703

8Jorgum NC C2705 a̲bill‑dun PRL20 10 a.admaJjs lac(Is ADO1 1

･1ql I

ITIAe

::I.::

rIII ILll 1LIII LLLll

TPrT CRT

TTT TTT

II(AArtIITC:C

*c TT rrT

AT

AA

;r!!‑i

TIAAAcAcACGllLIJceI7Ac

AAC AAC Ace QdG ANNACNCCNTTTTT6C ‑CNAAACACGCG‑CCNCGNAAC

‑10

CQC CC;C

GGC C6C CCG

a.dqlLEum Bdl a.adoLEFSCeI7Lis A 7CC 75703 BJotvum NCC2705

a. bJlidum PFIL2010 a,8nlnL?Illslacds ADO1 7

Ccnsensus

B

a.adolEZSCOhuS A TCC 7 5703

a.dmlium Bd1

8Jongum NCC2705 a.bJGhTr7 Pt?L2010 By8dm3IJs lacLLS ADO1 1

CmsbnSUB

a̲adolDSLHnLLls A TCC15703

&dentlum 8d7 BJorqum NCC2705

a.bd7dtln7 PRL2010 a̲brrmalJs lacrjs ADO7 7

Cmscnsus

a.edoloscEInL(s A TCC75703 a.denEium Bd1

8Jolgum NCC2705

8.bindum Pfa2010 8.&lVrmaJJs lacLEs ADO7 1

TARAQ TA#AG TA8AG TACAG

TAgAA

TACAG

･20l

BGgCPACCCTA AGE?C#Al= ^CCTA AGICF]Al=CCTAI

#GPCPIACCCTAJ B6gC&TCCCC

It>AACACQCG IAAACE}CACG L^AdCAAd[CG LTTE:QOC4CG

IILCl5l IIJTQ IbLOA JILcd

‑35

‑loo

TTGCe TGCt TGCC TGCT

TGAA

TGCC

#5GTAC:ACT)GJ ⁵⁴

Ef:;2:::!i;I

⁵⁵⁴⁵

gLt81'ACA6L1@b ^s4 P?ABCAA.L)qll ⁵⁵

;::: ::;::::::T,::E::S::I:E2:E:::!Gi:::::::

T6TT66TAAACAATR66C IC&IT6TT6GTAAACAA4GGC

111 111 110 110

GIcAJTGTTGGTTAACC^ALT;CCII ‑^GCAI(;TGI)AICCATCCGI ^1{1

GNCANTGT TGGTAAACAATGGCNTCAGTGN6CCNNANGCACGCGN

FIBS

AGGGA AG6GA

A6G6A AG G6A AG6GA

TilTA WTA WTA Fq‑A

F=TA TTA

‑120I

138 13$

138 138 134

:ttl I

cTTAtAAAATQaCA

cT TAIAGA^TQE:qQ

c TTi(A ^AAA EW.e@GIa]

CTTL Tt)

A^A^TGCQQ

AAAC&Cbgo

T TNAACCCCCNTNNNNGNAANAAANT6NGCGAAAACCCTTATAAAATGC6GGT

‑10

LggALcLTGAG

‑GAANCNTGC6

RBS ACW

ACG ACB ATl;

AC..

AGAAG A6AAG AGAAG AGAAG AGAAG A6AAG

ALVA AITA All7fA AICA AIBA

ATdTA

Tt

;;

;i

‑6D JW

L 1

cT1^TGA^CgT4qIA11TTdllL:; LAAqeIQL4TSyCCCCTG ^{1 16}

;iT̲iATr.^ACGTQGACiTCii

LTTI^TGAACACCE)qgLATIll

EIAAVCIOLATQTccccTG

IAAAE)IG)1‑T4QccccTG ^11t5116 JdAAQ]gIAIA

‑@gCCCC?6 ¹¹⁶ llTltTTGAACATMQqTAIMBIAATQIQIATT<CCCCTG ¹¹⁷

NTTNATGAACAT6G6GNTTNNGGNAATCNGNATGTCCCCTG

1

137 137 137 137 138

Fig. 1 8. Alignment of the upstream sequences of gap

(A)

and hup

(B)

genes. The _start codon, ^{proposed RBS,} ‑35 and ‑10 sites^were blocked.

0 U) a

‑a

〜 a

O

I+

0 JJ>

'j=

E a

=

O

4)

>

i;

‑q}

EE

0

Exponential‑phase Stationary‑phase

Fig. 19. Relative quantity

(RQ)

of catalase transcripts in B. longum 105‑A

bKKT427‑HkatE

^or

pKKT427‑GkatE)

^at exponential‑phase

(OD660

0f

‑0.6)

^and

stationary‑phase

(OD660

0f

‑1.0).

^{Data were normalized to} the chromosomal gap gene and analyzed by the 2‑AACT method. Values represent the fold _{change compared} ^with B.

longum 105‑A

bKKT427‑HkatE).

^The quantity of catalase transcript in B. longum 105‑A

Q)KKT427‑GkatE)

^{was 1.058‑fold than} the B. longum 105‑A

bKKT427‑HkatE)

at exponential‑phase, and the quantity of catalase transcript in B. longum 105‑A

bKKT427‑llkatE)

^{was 1.047‑fold than the B. longum 105‑A}

b)KKT427‑GkatE)

^at

stationary‑phase. Empty bar: B. longum 105‑A

bKKT427‑HkatE);

solid bar: B. longum 1 05‑A

bKKT427‑GkatE).

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

4.2.2 Translation

In prokaryotes, the promoter consists of2 short sequences ^atthe ‑10 and ‑35 positions

upstream &om the transcription start site. The length ofa RBS varies from 3 to 9 _nt, and the distance

bet&een

^{the RBS} and the initiation codon ranges from 5 to 13 _nt.

considering the promoter size, and the length of the RBS and spacer, ^we investigated the upstream ^areas of the 1,243 genes in which the interspaces of the coding region ^are longer than 50 _nt in B. longum NCC2705, which carries 1,727 hypothetical genes. We

counted the A, T, G, C &equency

(Fig.20).

^{The A + G contents are} very high in 18 _nt upstream of the initiation codon. We then counted the frequently appearing sequences 5 nt, 6 _nt and 7nt from

positions

^{‑1 to ‑18}

^(Fig.21).

^{Almost all high} &equency sequenceg

were complemented ^to the 3'‑end of B. longum 16S rRNA. AAGGAG showed the highest 6 _nt frequency which ^was 2.8‑fold compared ^to AGGAGG. We designed various RBSs with lengths from 5 nt to ll _nt, and with ^a 6 _nt spacer, according ^to the B. longum 16S rRNA 3'‑end

(5'‑...CACCUCCUUUCU ‑3');

^{AAGGAG was used as a core}

sequence

(Fig.22).

^These sequences ^were introduced into expression vectors and ^we assayed their catalase activities.

Quantitative

analysis of catalase activity ^was measured by the decrease of optical

density

^{at 240 nm as described. The} strain had the greatest activity when the RBS ^was AAGGAG

(6nt)

^{which matched} the statistical data

(Fig.23).

When the RBS was longer than 6 nt, the activity decreased in B. longum. This might be because the ribosome bound to mRNA ^so tightly that disturbed releasing the ribosome to start the translation. We also counted the 6 nt nucleotides with upstream fi.equencies

with the ^same manual in other

Bljidobacteria

species, including B. adolescentis ATCC15703, B.

bljidum

^PRL2010 and B. animalis subsp. lactis ADOll. AAGGAG ^was

the most ^common 6 nt nucleotide in all of the strains. These results indicated that

AAGGAG was the canonical RBS in

Blf2iobacteria. (7,

12, 16, 17, 31, 58, 62, 82,

83)

600

>

O

=q)

400

=

a ³⁰⁰

L

LL

200

‑27 ‑24 ‑21 ‑18 ‑15 ‑12 ^‑9 ‑6 ‑3 ⁰ Position

Fig. 20. The A, T, G, C &equency in the upstream ^areas of the genes with interspaces

of the coding region longer than 50 _nt in B. longum NCC2705.

The frequency of5 ^{nt, 6 nt,} and ^{7 nt} frompositions

‑1 ^to ‑18 AAG GA

AGGAG

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 75-83)