Molecular mechanism f6r coping with critical heat
and oxidative stresses in thermotolerant
Zン〃20〃20πα5〃ωゐ’1’5
（耐熱性Z画0〃ωπα5〃ωゐ’傭における限界熱ストレスと
酸化ストレスに対する対処分子）
Kannikar Charoensuk
2011
Molecular mechanism for coping with critical heat
and oxidative stresses in thermotolerant
2ン〃20〃20παM20ゐ’〃5
K紐nnikar Charoensuk
Graduate School of Medicine，
Yam訊guchi University，
2011
Gradu捌te School of Medicine
Yamaguchi University
Molecular mechanism血｝r coping with critic劉l heat
and oxidative stresses in thermotolerant
2ン〃20初0παM20ゐ’1∫5
Kannik裂r Ch紐roensuk
Doctor of Applied Molecular Bioscience
Graαuate School of Medicine，∼
Yamaguchi University，
2011
Contents
Page
1・ist of figures
i
I、ist of tables
ii
I、ist of abbreviations
Ch劉pter l
Introduction and review of litemtures
1．1General introduction
1．2Microorg劉nism genetic developments for bioethanol
iii
1
1
2
production
1．3The ethanologenic bacterium 2ン〃20〃20πα∫配oゐ漉∫and its
4
biotechnological potenti劉1
Ch紐pter 2
6
Molecular stmtegy fbr surviva豆訊t紐critical high tempemture in
恥C乃’ε7∫C配αCO〃
Abstmct
2．11ntroduction
2．2Material and methods
2．2．1Materials
2．2．2Bacterial strains and growth conditions
6
6
8
8
8
2．2．3Screening ofthe㎜o sensitive mutants
8
2．2．4Effbcts of glucose and MgCl2 and sensitivity to H202
2．2．5RT−PCR analysis
9
9
2．2．6Bioinfb㎜atics and phylogenetic analyses
11
2．2．7DNA chip analysis
11
2．3Results劉nd Discussion
2．3．1The㎜osensitive mutants and the㎜otolerant genes
11
11
2．3．2Effbcts of supplements and oxidative stress on growth of
16
the㎜osensitive mut㎜t strains
2．3．3Bioinfb㎜atics analysis ahd classi丘cation ofthe㎜otolerant
17
genes
2．3．4Possible acquisition of some thermotolerant genes by
22
horizontal gene transfbr
SupPlement Infb㎜ation
32
34
35
Ch紐pter 3
43
2．3．5Expressional change caused by heat shock at CHT
2．3．6Further consideration on mechanisms fbr survival at CHT
Molecular metabolism of thermotolemnce in eth3nologenic
themotolerant勿配o吻oπα∫鷹oゐ’Z∫5 apPlic紐ble fbr high temperature
fermentation
Abstract
43
Contents（cont．）
Page
3．2．1Materials
43
45
45
3．2．2Microorganisms
45
3．2．3Growth and ethanol pro duction of Zη20ろ∫1∫5 strains
46
3．2．4Corj ugation and strain construction
46
46
3．11ntroduction
3．2Materials紐nd methods
3．2．5Screening of the the㎜o sensitive mutant strain
3．2．6Effbct ofvarious stresses on growth ofthe㎜osensitive mutants
3．2．7DNA manipulation
3．2．81dentification of the transposon（Tn10）inserted site of
47
47
47
Z駕o励8the㎜o sensitive mutant genome by TAIL（The㎜al
Asy㎜etric Interlaced）PCR㎝d nucleotide sequence
analysis
3．2．9Comparison ofNucleotide Sequences
3．2．10Analytical procedures
3．2．11Statistics
3．3Results
3．3．1Comparative growth and ethanol production of
49
49
49
49
49
thermotolerant Zアηoゐ’1∫5 with the other type strains
3．3．2Screening ofthe㎜osensitive mutants and identi取the
51
the㎜otolerant genes ofZ励11∫5
3．3．31denti丘cation ofpossible the㎜otolerant genes
3．3．5Ef飴ct ofother stresses on the㎜otolerant mutants
3．4Discussion
Chapter 4
56
56
56
60
Physiological importance of cytochrome c peroxidase in
ethanologenic thermotolerant勿〃20〃20παMωゐ菰Zお
4．2．1Materials
60
60
62
62
4．2．2Bacterial strains， culture conditions and membrane
63
Abstract
4．11ntroduction
4．2Material 3nd methods
preparatlon
4．2．3DNA manipulation
65
4．2．4Halo assay
66
66
4．2．51ntracellular RO S level
Contents（cont．）
Page
4．2．6RT−PCR analysis
4．2．7ノ㎞alytical procedures
4．2．8Database search and computer analysis
4．2．9Statistics
4．3Results
4．3．1Structural characteri stics of ZmCytC
4．3．2Effbcts of disruption or increased expression of Z加（ツ∫C on
67
67
68
68
69
69
70
morphology， growth and ethanol production
4．3．3Sensitivity to H2020f a disrupted mutant of Z〃z（γC
4．3．4Expression of genes presumably related to oxidative stress
74
76
response and effbct of a disrupted mutation of Z加のノ∫C on
their expression
4．3．5Peroxidase activity of ZmCytC and its possible electron
78
donor
4．4Discussion
80
Refbrences
82
Ac㎞owledgements
101
Summary
Summary（Japanese）
Ijst of publications
102
104
106
正ist omgures
Page
Chapter l
Figure 1．1
Figure 1．2
The morphology of Z吻ob／1∫5
M句or carbohydrate pathway in Z加oろ∫1∫5
4
5
Chapter 2
Figure S 1
Gro舳ofthe㎜osensitive mutants in LB liquid culture
39
at diffbrent temperatures
Figure S2
Gene organizations around genes having either an essential
40
gene or a the㎜otolerant gene as a j ust downstre㎜gene
Figure S3
Testing ofpossible polar effbcts l）y the角ψinsertion
41
Figure S4
Ef琵cts of addition of glucose and MgC12 and sensitivity
42
toH202
Figure S5
Do㎜一regulated genes fbr ribosomal proteins
42
Chapter 3
Figure
Phylogenic tree analysis of 16srRNA ofthe
50
3．1
Figure
3．2
Figure
3．3
Figure
3．4
the㎜otolerant Z御oゐ∫1∫∫TISTR 548 and other type
strains
Comparison of growth abilities of the Z吻oゐ∫1∫5 TISTR
548with other type strains under stresses condition
50
Growth and ethanol production of Z吻ob∫1∫5 strains in
51
YPD medi㎜㎜der static condition．
Relative growth of the㎜osensitive mutant strains with
52
its parental strain
Chapter 4
Figure 4．1
Alignment of deduced amino acid sequences of
70
Figure 4．2
ZmCytC and other bCCPs．
Growth curves and moΦhology of Z加（γC mutant and
73
parental strains under a shaking condition and effbcts
ofZ加（ツ∫C clone．
Figure 4．3
Effbct of disruption of Z〃2（ッ’C on glucose utilization
74
and ethanol production
Figure 4．4
Effbct ofdisruption or increased expression of Z加のノ’C
on sensitivity to exogenous H202
i
75
I」ist of t紐bles
Page
Chapter l
Table 1．1
Genetic engineered microorganism fbr utilization of usefhl
3
materials fbr ethanol production．
Chapter 2
Table 2．1
RT−PCR primers used in this study
10
Table 2．2
The㎜otolerant genes identi丘ed in this study
13
Table 2．3
Distribution ofthe㎜otolerant genes in group D in various
19
bacteria
Table 2．4
Distribution ofther士notolerant genes in various bacteria．
Table 2．5
Distribution ofthe㎜otolerant genes in group B in vadous
24
28
bacteria
Table 2．6
Gene s significantly up−regulated and down−regulated at
Table 2．7
Essential genes significantly up−regulated and down−
CHT
33
34
regulated at CHT
Chapter 3
Table 3．1
Reaction parameters of TAIL−PCR fbr amplification of the
48
丘anking region of Tn10 inse宜ed in the the㎜osensitive
mutant strains of Z溺ooわ’1’3 TISTR 548 with the modified
丘om previous report（Liu and Whitter，1995）
Table 3．2
Functional distribution of Zη20ゐ∫1∫3 the㎜otolerant genes
53
Ch紐pter 4
Table 4．1
Bacterial strains and plasmids used in this study 63
Table 4．2
Primers used in this study 64
Table 4．3
Peroxidase and oxidase activities in membrane fヒactions 79
丘om the parental and∠Z吻のノ∫C cells
ii
List of abbreviations
bp
BSA
°
C
Base pair
Bovine se㎜alb㎜in
Degree Celsius
DNA
Deoxyribinecleic acid
et al．
and others
9
gram（S）
h
hour（s）
kb
ldlobase（s）
kDa
kilodalton（s）
Km
Kanamycin
L
Liter（S）
LB
Luria−Be価medi㎜
M
Molar
mg
milligr㎜（s）
min
minute（S）
耐
millimolar
μ9
microgram（S）
μ1
miCrOliter（S）
μM
miCrOmOlar（S）
iii
1・ist of abbreviations（cont．）
NAD＋
oxidized nicotnam．ide adenine dinecleotide
NADP＋
oxidized nicotn㎝ide adenine dinecleotide phosphate
NADH
reduceded nicotinamide adenine dinecleotide
NADPH
reduceded nicotinarnide adenine dinecleotide phosphate
㎜
nanometer（S）
PCR
Plymerase chain reaction
rpm
revolutions per minute
SDS
Sodi㎜dodecyl su1魚te
SDS−PAGE
Sodi㎜dodecyl sul魚te polyacWlamide gel electrophresis
YPD
Yeast Peptone Dextrose
％
percent
iv
Chapter 1
Intmduction anαreview of literatures
1．1General introduction
The world population on Earth of July 2008 is estimated to be just over 6．68
billion expected to reach nearly g billion by the year 2042． The additional people will be
almo st all in poorer developing countries． A rapidly growing world population required
more飴od（h杭p：／／w四．co㎜ondre㎜s．org／news2000／0523−01．htm）． Global demand
fbr cereals is pr（jected to increase by 40％． Meat demand is expected to increase by
58％and demand fbr roots and tubers by 37％． The most of this ibcrease coming丘om
the developing world． Demand fbr f㎞its， vegetables and seasonings as well as nonfbod
farm products will also rise． But fUture increases in fbod productiqn and new land to
expand the agricultural base are likely to be more dif丘cult because a complex range of
enviro㎜ental and social魚ctors．
In too many fbods production regions of the world have severely depleted
because supplies of oil and gas are essential to modem agriculture tec㎞iques， a fall in
global oil supPlies could cause spiking fbod prices and unprecedented famine in the
coming decades． Nowadays cmde oil prices behave much as㎝y other co㎜odi騨ith
wide price swings in times of shortage or oversupply． The cnlde oil price cycle may
extend over several years responding to changes in demand as well as OPEC and
non−OPEC supply． In 1983 crude oil price was about 30 US． Dollar per barrel during 25
years the price raise dr㎜atically with 400％at 140 US． per baエrel． Because qf world
high dependence of most modem industrial transport， agricultural and industrial systems
on the relative low cost and high availability of oil will cause the oil−peak production
decline and possible severe continue increases in the price of oil to have negative
implications fbr the global economy （http：／／en．wikipedia．org／wiki／Peak＿oil）．
The altemative energy comes to substitute fbr oil． In 2006， about 18％of global final
ener留cons㎜ption c㎜e丘om renewable source， with 13％coming丘om廿aditional
biomass． Hydropower was the next largest renewable source， providing 3％， fbllowed
by hot water／heating， which contributed 1．3％． Modem tec㎞ologies， such as
一
1一
geothe㎜al， wind， solar， and ocean energy together provided some O．8％of丘nal energy
cons㎜ption（h廿p：／／www．i−sis．or g．面SustainabIeWorldI㎡tiativeF．php）．
Bio−fUel as a member of renewable energy which uses the energy contained in
organic matter−crops such as sugarcane or corn include wheat crops， waste straw，
willow and popular trees， sawdust， reed canary grass， cord grasses， Jerusalem artichoke，
myscanthus and sorgh㎜plants． To produce ethano1，㎝altemative to飾ssil−based fUels
like petrol．
Ethanol or ethyl alcohol（C2H50H）is a clear colorless liquid；it is biodegradable，
10w in toxiciW㎝d causes li賃le enviro㎜ental pollution if spilt． Eth㎝01 b㎜s to
produce carbon dioxide and water． Ethanol is a high octane fUel and has replaced lead
as an octane enhancer in petrol． By blending ethanol with gasoline and oxygenate the
釦el mixture so it b㎜s more completely and reduces polluting emissions． Ethano1釦el
blends are widely sold in the United States and still increase around the world
（http：／／www．esru．strath．ac．uk／EandE／Web＿sites／02−03／biofUels／what＿bioethano1．htm）．
The production of bioethanol around the world was 411）illion liters in 2004． The largest
producers in the world are Brazil with 37％， US with 33％and Asia with 14％． Below is
adescription of EUラs fbcus on bioethano1（http：／／www．biogasol．dk／3me4．htm）． Global
demand to approach 90 million metric tons in 2011−Demand fbr biofUels will expand
almost 20％per year through 2011 to 92 million metric tons． On another hand the
World Bank reported cause of increased bio fUel production has contributed to the rise
in fbod price． So increase the efficiency of bioethanol production processes such as used
the ethanologenic the㎜otolerant microorganism is expected to become one of the
economical next−generation fb㎜entation tec㎞ologies．
1・2Microorganism genetic developments for bioeth紐nol production
The impo貢ance of yeast cell physiology in alcohol飴㎜entations has been
emphasized by several researches such as fbr艶㎜entations of pentose by S oθFεv∫51αθ
［K6tter et al．，1990；Tantirungk勾et al．，1993］and Z脚oわ∫1∫3［Deanda et al．，1995］， fbr
角㎜entations of high−gravi取cereal［Thomas and Ingledew，1992］and fbr
飴㎜entations of sugar cane molasses［Walkerθ’α1．，1996］． Genetic engineering
micro−organisms fbr utilization of usefUl materials fbr ethanol production are°noted in
Table 1．1．
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1．3The ethanologenic b劉cterium Z 1ηoわ’1なand its biotechnological potentia1
Bα（3旋∼7’α；
1）君0’θ0ゐα0’ε7ゴα；
∠霊加乃qρ召0’θ0ゐOlαθπ0；
勘乃加go〃20ηα4α1θ5・；
5ン痂η90〃20ηα6劾086Zθ；
Zン〃20〃20η05；
2ン〃20刑oηα3〃20わ’1’5・
Figure 1．1 Morphology ofみ〃20脚ηα5溺o励5
The genus Zソη20〃20ηα5 consists of large， gram−negative rod（2−610ng and 1−4
wide）， which cany out a vigorous鉛㎜entation of sugars to homo−ethanol． Although not
all strains are motile， if motility occurs， it is by lophotrichous flagella．身配o脚ηα5 is a
co㎜on organism involved in alcoholic色㎜entation of various pl㎝t saps， and in
mally tropical aleas of South and Central America， Af｝ica， and Asia， it occupies a
position similar to that of S cθアθv’5∫αε（yeast）in North America and Europe．
〃刑o脚ηα3is involved the alcoholic飴㎜entation of agave in Mexico， and palm sap in
many tropical areas． It also carries out of ethanol fb㎜entation ffom sugarcane j uice and
honey． Although 2ン初o〃70ηα5 is rarely the sole organism involved in these alcoholic
免㎜entations， it is o負en the dominant organism and is probably response fbr the
production of most of the ethanol in these beverages．2ン刑o配oηα5 is also responsible fbr
spoilage of丘uit juices such as apPle cider and perry． It also may be the constituent of
the bacterial flora of spoiled beer and may be responsible fbr the production in beer of
unpleasant odor of rotten apples．身溺oη20ηα5 resembles most closely the acetic acid
bacteria， specifically Glzκoηoろαo∫θγbecause of its polar flagellation， and it is often
fb㎜d in nature associated with the acetic bacteria． This is of interest because
み刑o脚ηα5色㎜ents glucose to ethanol， whereas the acetic acid bacteria oxidize
ethanol to acetic acid． Thus， the acetic acid bacteria may depend on the activity of
2：ソ〃20〃20ηα3fbr the production of their growth substrate， ethanol． Like the acetic acid
bacteria， Z吻o脚ηα3 is quite tolerant of low pH． Unlike yeast， which免㎜ents glucose
to ethanol via the Emden−Meyerhof（glycolytic）pathway， Z卿o吻oηα5 employs the
Enter−Doudoroff pathway． This pathway is active in many pseudomonads as a means of
catabolizing glucose．
身η20刑oη05is of interest to the ethanol industry because it shows higher rates of
glucose uptake and ethanol production and gives a higher yield of ethanol than many
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types of yeast．2：ソ〃20〃20η03 is also rather tolerant of high ethanol concentrations（up to
10％）but is not quite as tolerant as some of the best yeast strains， which can grow to
12−15％ethanol． However， the魚ct th翫加o吻o鷹is a Gr㎜一neg翫ive bacteri㎜and
can thus be readily manipulated genetically makes it an attractive candidate fbr use by
ethanol production industries．
We report the complete genome s閃uence of Z襯oゐ∫1∫5 ZM4（ATCC31821）， an
ethanologenic microorganism of interest fbr the production of fUel ethanol． The genome
consists of 2，056，416 base pairs fb㎜ing a circular chromosome with 1，9980pen
reading ffames（ORFs）and three ribosomal RNA transcription u㎡ts． The genome lacks
recognizable genes fbr 6−pho sphofhlctokinase， an essential er騨e in the
Embden−MeyerhofP㎜as pathway， and fbr two enzymes in the tricarboxylic acid
cycle， the 2−oxoglutarate dehydrogenase complex and malate dehydrogenase， so glucose
can be metabolized only by the Entner−Doudoroff pathwaひWhole genome microarrays
were used fbr genomic comparisons with the Z配oゐ∫1∫5 type strain ZM 1（ATCC10988）
revealing that 54 0RF s predicted to encode fbr transport and secretary proteins，
transcriptional regulators and oxidoreductase in the ZM4 strain were absent丘om ZM 1．
Most of these ORFs were also fbund to be actively transcribed in association with
ethanol production by ZM4［Seo et al．，2007］． ・
Levan
霊謡鳳n。
盤誌璽聖器．
無酩艦鵬ic
Ghlconat
Sorbito鳳
Caπier
Caロ重er
鮒 4G且署惣 。詣㎞、
ADH：alcohol dehydrogonases I and II；
EDA：2−keto−3儘deQxy一εlucσnate−alddase；
EDD：6−phospho創ucαロate dehydra憩sc；
∼襲 幸
6璽P−（｝1uconoiactone
PG乙
ENO：enolase；
FRK：Fructokhlase；
GAP：glyceraldchydc 3−phosphate
dehydrogcnasc；
6− luconatc
Erythrose−4 班）D
騨鷲炉澱曳琳
GFOR：glucose−fnlctose oxidQreductase；
vate
GLF二富1ucose facilita勧or；
義轟、
今・・粥
GLK：謬u。o㎞ase；
GNL：ε1uconolact㎝ase；
PDC
GNT K：91uconate khlase；
INV B：i且vc：tase B；
2樗G㎏鰐
KDPG：2−ketoρ3−deo区y6−pho霞pho画u◎onate；
LEV U：1evansucraso；
Phosphoenolpymvate
識照
C
inz」脚屍酌
Abbreviadons
Fructose−5−P− G五ucose−6−P
寸z陀F
1，3−Di4）−Glycerate
Majorcarbohydr劉te pathways
ME：malic enzyme；
PDC：pyruvate decalboxylasc；
PDHC：pyruvate ddhydrogeaase oo風plex；
at Acet・ld・hyd・＋CO 2
衆誌羅吻↑↓−
PGI：phospho創uco iso皿o窮竃so；
PGK：phospho曾yc￠rate kinas￠；
Oxald cetate E出ano且
Citrate
PGL：phosphogLuconQla6toaase；
孟幽＿
PPC：phosphoθηoφyruva加carboxylaSe；
PYK：pyruvate k㎞se；
TKT：廿ansketolasc；
M撫 温、離
PGM：phospho蜘cαomutase；
Fu㎞arate
ZVのF：9【ucosc 6−pho領ha1雄dehydmgenase．．
、 ’
ヘ ノ
’、 Suc6重ny1℃oA
Succmate−＿一一’一鴨
Figure 1．2 M句or carbohydrate pathway in Z〃励’1∫3
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Chapter 2
Molecul劉r strategy fbr survi剛紐t a critic紐l high temper蜘re in
E5c乃’87’c〃’αω〃
Abstract
The molecular mechanism supporting survival at a critical high temperature
（CHT）in E． co1∫was investigated． Genome−wide screening with a single−gene㎞ockout
library provided a list of genes indispensable fbr gro舳at 47°C， called the㎜otolerant
genes． Genes fbr which expression was affbcted by exposure to CHT were identified by
DNA chip analysis． Unexpectedly， the飴㎜er contents did not overlap with the la廿er
except fbr伽α1 and伽αK， indicating that a specific set of non−heat shock genes is
required fbr the organism to survive under such a severe condition． More than half of
the mutants of the the㎜otolerant genes were鉛㎜d to be sensitive to H202 at 30°C，
suggesting that the mechanism of the㎜otolerance脚ially overlaps with that of
oxidative stress resistance． Their encoded enzymes or proteins are related to outer
membrane organization， DNA double−strand break repair， tRNA modification， protein
quality control， translation control or cell division． DNA chip analyses of essential
genes suggest that many of the genes encoding ribosomal proteins are down−regulated
at CHT． Bioi㎡brmatics analysis and comparison with the genomic infb㎜ation of other
microbes suggest that E． oo1∫possesses several systems fbr s㎜ival at CHT． This
analysis allows us to speculate that a lipopolysaccharide biosynthesis system fbr outer
membrane organization and a sulfUr−relay system fbr tRNA modi丘cation have been
acquired by horizontal gene transfbr．
2．11ntroduction
Responses of E5c乃θγ∫c乃∫αco1∫to high temperatures have been extensively
investigated， though previous studies have mainly fbcused on the response to a
tempera傭e up−shi食訂o㎜d 42℃， a response㎞o㎜as a heat shock response（HSR）to
induce the expression of a set of proteins， heat−shock proteins（HSPs）［Morimoto et al．，
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1996］．The fact that many HSPs are conserved㎜ong species indicates that the actions
of HSRs are the fUnd㎜entally and physioIogically important mechanisms in living
organisms［Boorstein et al．，1994；Gupta，1995］． HSPs play crucial roles not only in the
rescue or removal of proteins damaged by enviro㎜ental stresses， including heat stress
and salt stress， but also in the int血sic fblding of proteins㎜der no㎜al gro舳
conditions［Yura et al．，2000］．
It has been sho㎜taht 384 genes are up−regulated by short−time exposure to a
temperature of 43°C as a heat shock in E． co1∫［Gunasekera et al．，2008］， and these genes
may be directly or indirectly induced by the treatment． The directly induced genes
encode HSPs， including the main cellular chaperone machineries of GroEL and DnaK，
ATP−dependent proteases of Lon， HsIUV， Clp and FtsH（HfB）， periplasmic protease
DegP， and other proteins involved in protein fblding， refblding， quality control and
degradation［Narberhaus et a1．，1998］． HSPs are under complex regulations and are
divided into several regulatory groups by their m句or stimulons［Erickson and Gross，
1989］．The control of their expression， however， is highly variable㎜ong orga㎡sms
and even among various bacteria［Raina et al．，1995］．
One of the control elements fb㎜d in Gram−negative bacteria is a heat shock
sigma factor that regulates transcription of the m句or HSPs． HSR in E． oo1∫is generally
mediated by altemative sigma factors， sigma 32 and sigma 24［Yura et a1．，2000；
Erickson and Gross，1989；Raina et al．，1995］． Transcription of theぞρoH gene fbr sigma
32is induced at elevated temperature via the action of sigma 24［Erickson and Gross，
1989］．Sigma 24， which is inactive under non−stress conditions by interaction with
anti−sigma factor， is activated by misfblding of outer membrane or periplasmic proteins
and by stresses including heat shock［Alba et al．，2002］． Both sigma factors are fUrther
regulated at the translation level and or at the posttranslational level． The factor sigma
24is in part regulated by a cognate small RNA， and sigma 32 synthesis is regulated by
stnlctural change of its own mRNA molecules serving as a cellular the㎜ometer and its
activity modulated by phosphorylation［Morita et al．，1999；Klein et al．，2003］． Other
microorganisms， on the other hand， appear to possess diverged regulatory mechanisms
［Helmann and Chamberlin 1988］．
There is no i㎡b㎜ation on the molecular mechanisms of response to and
survival at a critical high temperature（CHT）in orga㎡sms， probably due to the limited
expe㎡mental procedures． Developments of a single−gene㎞ockout library and DNA
chip analysis have encouraged us to perfb㎜agenome−wide investig翫ion of responses
in organisms under extreme conditions． Since several mesophilic bacteria including E．
co1∫can grow and survive at high temperatures compared to other mesophilic bacteria，
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they are assumed to have acquired the potential fbr the㎜otolerance during their
evolution． In this study， we utilized・new procedures fbr the first time to obtain
infb㎜ation on the molecular mechanisms related to the㎜otolerance in E． co1ゴat CHT．
Screening of the㎜osensitive mutants at CHT and in飴㎜atics analysis of the
corresponding genes revealed pathways or factors indispensable fbr survival at CHT．
For essential genes， their possible involvement in the response to CHT was exa再nined
by DNA chip analysis． Based on the results， we propose novel molecular mechanisms
fbr survival at CHT in E． co1ノ．
2．2Materials and Methods
2．2．1Materials
Oligonucleotide primers fbr polymerase chain reaction（PCR）were purchased
丘om FASMAC Co， Ltd（Atsugi， Japan）． Other chemicals were all of analytical grade．
2．2．2Bacterial strains and growtll conditions
Strains used in this study were derivatives of E． co1’K−12． W3110（IN
（77ηD−〃ηE），ηフ乃一1），BW25113（〃η、B3，∠4α02ひ4787，乃34R514，∠＠α1祖切567，
∠帥α甜1刀568，吻一1）［Datsenko and Wanner，2000］and mutants of BW25113 in the
Keio collection as a single−gene㎞ockout library［Baba et al．，2006］were gro㎜on
plates or in liquid of modified Luria−Berta㎡（LB）medium（1％Bactotryptone，0．5％
yeast extract， and O．5％NaCl）at 37°C，45°C or 47°C fbr appropriate times．
2．2．3Screening of thermosensitive mutants
The Keio collection consisting of 3，908 mutant strains was used fbr screening．
In the lst screening， mutant strains were gro㎜on LB plates at 30°C ovemight． A
colony of each strain was patched on LB plates and incubated at 47°C fbr 48 h to find
sensitive strains． The sensitive strains were su切ected to the 2nd screening of spotting
tests on plates． Cells were cult皿ed in LB medi㎜鉛r 18 h and then diluted with LB
medi㎜to a両ust turbidi旬to OD6000f O．5，0．05 and O．005． The diluted samples（10μ1）
were spo廿ed on LB plates and incubated at 47°C fbr 48 h． The the㎜osensitive strains
selected by the 2nd screer血g were su切ected to the 3「d screening in liquid culture． After
8−hpreculture， cells were diluted to a turbidity corresponding to OD6000f O．1 and
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inoculated into LB medi㎜at the且nal OD6000f O．001．Samples were then incubated at
47°C飴r18 h under a shaking condition． The㎜osensiti∀ity was dete㎜ined by
measuring OD600． The㎜osensitive strains were de丘ned to be＜0．1 at OD600． The
experiments were perfb㎜ed three times， and the results were con丘㎜ed to be
reproducible．
2．2．4Ef6ects of glucose and MgC12 and sensitivity to H202
To ex㎜ine effbcts of supplements， glucose（0．5％（w／v））or MgCl2（20 mM）
was added to the LB liquid culture． After 8−h preculture， cells were diluted to a turbidity
coπesponding to OD6000f O．1 and inoculated into LB medi㎜with or without the
supplement at the final OD6000f O．001． Samples were then incubated at 47°C fbr 18 h
㎜der a shaking condition． After 18 h， turbidity at OD600 was measured． To test the
sensitivi砂to oxidative stress， H202 was added to the cul血e medi㎜at the且nal
concentration of O．5 mM． After 8−h preculture， cells were diluted to a turbidity
coπesponding to OD6000f O．1 and inocul誠ed into LB medi㎜with or without H202翫
the丘na10D6000f O．001． Samples were then incubated at 30°C fbr 8 h㎜der a sh泌dng
condition． The expehments were per鉛㎜ed three times， and the results were con五㎜ed
to be reproducible．
2．2．5RT−PCR analysis
Cul加res were gro㎜in LB medi㎜at 37°C until the exponential phase， and
then the temperature was up−shifted to 47°C and incubation was continued fbr 8 min．
Total照A was i㎜ediately prep肛ed丘om the heat−stressed cells by the hot phenol
method［Aiba et al．，1981］． RT−PCR analysis was perfb㎜ed using an mRNA−selective
RT−PCR kit（T臨IU BIO Inc， Otsu， Ja脚）to ex㎜ine the expression of i㎜ediate
do㎜stre㎜genes of disrupted genes as described previously［Tsunedomi et al．，2003］．
The primer set used fbr each gene is sho㎜in Table 2．1． The RT reaction was carried
out at 42°C fbr 15min，85°C fbr l min，45°C fbr l min and extension at 72°C fbr 2 min
using the two specific primers fbr each gene． After the completion of 15，20，25 and 30
cycles， the PCR products were analyzed by O．9％agarose gel electrophoresis and
stained with ethidium bromide． The relative amounts of RT−PCR products on the gel
were compared by measuring the band density after the color of the image taken had
been reversed using a model GS−700㎞aging Densitometer（Bio−Rad Laboratories， Inc，
Tokyo， Japan）［Nitta et al．，2000］．
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Table 2．1 RT−PCR primers used in this study．
N㎜e
Sequence
aceF−5’
5’−GAGATCCTGGTCAAAGT−3’
5’−GCCAGCAAACGGAGCCG−3’
5’−AACTCAGGTCGTGGTAC−3’
5’−AACGGCAGTTGGATCGG−3’
5’−CGATGGGCGATGTTCTC−3’
5’−CATCTGTAGGCAGGTTC−3’
5’−ACATGATGATGTCGCAA−3’
5’−TTGTTATGGCCGCAGTT−3’
5’−GTCTAACTCGCACCCGA−3’
5’−CCGTTCTCTTCCGCTTC−3’
5’−GAGGTCGTCGCGATCTCA−3’
5’−CGGTTGGTTTAATGACGCA−3’
5’−AAGATATCGAACAGCCG−3’
5’−TTCACCGCATTGGACAG−3’
5’−CAGTTAACCAGCTGGTA−3’
5’−CTTCACGCCATACTTGG−3’
5り一CACATTACATCGCTCAC−3り
5’−TCACCAGGCCATCTGGC−3’
5’−CTACTGCACCTCATGGT−3’
5’−TCGTAGTTGGCGAGTTC−3’
5’−TTGCCATCGTGGTGACC−3’
5’−GAGCGAGGCTTCCGCCA−3’
5’−CAGCGGAAGAGCGTGAA−3’
5’−GAAGAATCCCTGGCGCA−3’
5ラ。CCGTATAGCAGGCATTA−3’
5’−TTGATCGGTTTGCGCGG−3’
5’−GGCAAAGGCACCAATTCG−3’
5’−TAGTGATGCGGAAACCTG−3’
5’−ACGTTATTTCCGTCGTCG−3’
5’−GATCAGTGTACGGCAGCA−3’
aceF−3’
lpd−5’
lpd−3’
rfac−5’
rfac−3’
rfaF−5’
r飴F−3り
der−5’
der−3’
tolR−5’
to1R−3層
valS−5’
valS−3’
rpsL−5’
rpsL−3’
yheL−5’
yheL−3’
yheM−5’
yheM−3’
yheN−5’
yheN−3’
dnaJ−5’
dnaJ−3’
rpsM−5’
rpsM−3’
rpsK−5，
ΦsK−3’
rpsR−5，
rpsR−3’
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2．2．6Bioinform紐tics and phylogenetic an劉lyses
Bioinfb㎜atics analysis was mainly perfb㎜ed according to the instmctions of the
KEGG site （http：／／www．genome．j p／kegg−birゾshow＿organism？ menu」ype＝pathway
maps＆org＝e（」）． Datal）ases of DDBJ and GenBank were also used．
2．2．7DNA chip紐nalysis
W3110 cultures were gro㎜in LB medi㎜at 37°C mtil the exponential phase，
and then the temperature was up−shifted to 47°C and incubation was continued fbr 8
min． A control culture was incubated in parallel at 37°C fbr 8 min． Total RNA was
i㎜ediately prep肛ed丘om the heat−stressed cells by the hot phenol method
［Tsunedomi et al．，2003】． Preparation of cDNA，丘agmentation and the end−labeling of
DNA丘agments were perfb㎜ed according to the instruction manual丘om Af恥etdx．
The ENZO Bioaπay te㎜inal labeling kit（Enzo Li飴Sciences， Inc， New York， USA）
was used to end−label DNA丘agments． DNA hybridization， data capture and analyses
were perfb㎜ed as deschbed in the protocol supplied by Af馳etdx and GCOS
software（Affymetrix， Inc， Califbrnia， USA）． Two independent experiments were
perfb㎜ed and fbur data sets（two d翫a sets at 37°C：37°C−1 and 37°C−2， two data sets at
47°C：47°C−1and 47°C−2）per gene were obtained． The expression ratio used here
indicates the average of the ratios obtained in the two independent experiments． Spots
with a significantly lower（＜0．50；i．e．， a negative fbld diffbrence）or higher（2＞；i．e．， a
positive fbld diffbrence）fluorescence ratio of the heated sample to the control sample
were considered to represent a real significant diffbrence． Physiological fUnction and
且mctional classification of the genes were derived丘om the Genobase database
（http：／／ecoli．aist−nara．ac．j p／GB 6／search．j sp）． Array data were submitted to AlrayExpress
㎝dthe accession n㎜ber’will be available on the web site
（http：／／www．ebi．ac．uk／arrayexpress／）．
2．3Results and Discussion
2．3．1Thermosensitive mutants and thermotolerant genes
hl order to identifシgenes required fbr survival at CHT in E． oo1ノ， we screened
fbr the㎜osensitive mutants丘om a single−gene㎞ockout libr飢y［Baba et a1．，2006）］，
which had been constructed according to the one−step gene disruption method with an
一
11一
￠ρ乃cassette［Datsenko and W㎜er，2000］and fbr which each construct had been
con丘㎜ed extensively［Yamamoto et al．，2009］． In the disrupted gene of each mutant
strain， the region between the l st codon and the last 6 codons was displaced with the￠ρ乃
cassette， so that most of the coding region of the gene was deleted． Our experiments
indicated that the parental strain used fbr construction of the disrupted library is able to
grow at temperatures up to 47°C， this temperature thus being its CHT．
Afier three successive screening steps of the library， including 3，908
disrupted−mutant strains，51 strains were fb㎜d to be sensitive to CHT． Their growth
curves at 37°C，45°C and 46°C were then compared to those of the parental strain
（Figure S 1）． The growth profiles suggest that most mutants selected are significantly
sensitive to 46°C and some even to 45°C． Such a disrupted gene responsible fbr the
the㎜osensitive phenotype was designated as a the㎜otolerant gene（Table 2．2）．
一
12一
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The gene organization generated by construction of the disrupted mutants might
give rise to a polar effbct of the inserted￠ρ乃gene on transcription of downstream genes
that are intrinsically transcribed by read−through丘om the promoter or the region
up stream of the disrupted gene． Such an organization was fbund in 420f the 51 mutants．
Sensitivity was not due to a polar effbct in 290f those 42 mutants because disruption of
genes just downstre㎜丘om the disrupted gene by the same method caused no
the㎜o sensitive phenotype． The remaining 13 mutants have either an essential gene or a
the㎜otoler㎝t gene as㎝i㎜ediate do㎜stre㎜gene（Figure S2）． Their possible polar
effbcts were thus tested by RT−PCR with total RNA prepared丘om cells exposed to a
temperature of 37°C or 47°C（Figure S 3）． The results suggest that the transcription level
of the i㎜ediate do㎜stre㎜gene in the mu伽t was almost the s㎜e as that in the
parent in all cases except fbr the cases of mutants ofαcθF，’01g，4加K and脚F． Most
of these do㎜stream genes would thus have their o㎜promoters or the transcription
level by read−through would be nearly the same as that of the￠助promoter． However，
the transcription levels of加61，’01R and伽αノlocated downstreε㎜ofαoθF，’01g and
伽oκ，respectively， were increased and the level ofκρ5．R located downstream of刑ρ3F
was decreased compared to those of the parental strain at both temperatures． Although
the expressional alteration of the 4 genes was nearly the s㎜e at both temperatures，
growth of the corresponding mutant strains at 37°C was not sigr直ficantly changed丘om
that o f the parental strain． Taken together， the results suggest that the the㎜otolerant
phenotype in the 51 mutants is due to disruption of the targeted gene and not due to a
polar ef色ct on its do㎜stream genes． Out of the 51 the㎜otolerant genes，8genes，
のノ詔，漉9P，6加α巧 6加02く 伽α9，ηろρ五 ψD and 吻C， had been reported as genes
supporting grow h at a high temperature．［Wall et al．，1992；Raina and Georgopoulos
1991；Klein et al．，2009；Adams and West 1996；Tomoyasu et al．，1998；Lipinska et a1．，
1990；Ohara et al．，1999］， and thus we newly identi旦ed 43 the㎜otolerant genes in this
organlsm・
2．3．2E価ects of supplements and oxidative stress on growth of thermosensitive
mutant stmins
Since LB was utilized as a medium鉛r the screening of止e㎜osensitive mutants，
limitation of carbon source might cause sensitiveness to CHT． We thus examined the
ef飴ct of glucose as a supplement fbr gro舳of the the㎜osensitive mutant strains
（Table 2．2， Figure S4）． We also tested the effbct of MgCl2 because Mg2＋somehow
protects against cell d㎜age under stress conditions［Kabir et al．，2005；Noor et al．，
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2009］．The growth of 20 and 37 mutants was improved at CHT by the addition of O．5％
glucose and 20 mM MgCl2， respectively． The growth of sixteen mutant strains was
improved by supplementation of not only glucose but also MgCl2．
Next， the effbct of exogenous oxidative stress on the the㎜osensitive mutant
strains was tested since a higher temperature causes more oxidative stress（Noorθ∫α1，
2009；unpublished data）． We exposed the㎜．osensitive mutant strains to O．5 mM H202 in
LB liquid medi㎜at 30°C． Twen取一nine mutants were鉛und to be sensitive to H202
（Table 2．2， Figure S4）， corresponding to approximately 60％of the the㎜o sensitive
mutants． Moreover， out of the 10the㎜osensitive mutants fbr which glucose and MgCl2
supplementation had no effbct，9mutants showed sensitivity to H202． These results
suggest that the mechanism of the㎜otolerance at CHT pa匠ially overlaps with th翫of
oxidative stress resistance．
2．3．3Bioinf6rmatics紐nalysis and classi血cation of thermotolerant genes
To understand the molecular mechanism of」E． oo1∫survival at CHT，
bioi㎡b㎜atics analysis with various public d翫abases including the KEGG PATHWAY
database was perfb㎜ed． Out of the 51 the㎜otolerant genes，29 genes were
successfUlly mapped on E． oo1∫pathways in the KEGG PATHWAY database．
Interestingly， many genes were fb㎜d to be involved in the same metabolic pathway，
suggesting that the orgar直sm possesses indispensable pathways at CHT． The remaining
19genes except fbr 3 un㎞own genes were extensively analyzed by using the DDBJ or
GenBahk database． On the basis of results of these analyses and the effbcts of the
supplements， the 51 the㎜otolerant genes were classi且ed into 7 groups（Table 2．2）．
Group A consists of genes concemed with energy metabolism fbr production of
ATP． The gene products ofαcθ」E，αcε」配功4 andαoん4 are mapped in the pyruvate
metabolism pathway丘om pyruvate to acetyl CoA［Haydon et al．，1993；Lyngstadaas et
al．，1995；Cassey et al．，1988；Barak et al．，1998］and that ofηワθis located in the
pento se pho sphate pathway．（ッ詔andア乃cB encode subunits of cyto chrome 4 te㎜inal
oxidase， which generates the membrane potential responsible fbr ATP synthesis［Wall
et al．，1992；Mogi et al．，2006］．1脚， which encodes LipA to produce lipoate required
fbr pyruvate dehydrogenase reaction， also contributes to pynlvate metabolism［Reed
and Cronan，1993］． Based on the results showing that disrupted mutations of these
genes caused a the㎜osensitive pheno卿e， we assumed that the cells require more ATP
at a higher tempera加re． This ass㎜ption was suppo丘ed by the且nding that the
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phenotype of most mutants in this group was partially suppressed by the addition of
glucose（Table 2．2， Figure S4）．
Group B consists of genes related to biosynthesis of the cell wall or organization
of the outer membrane． The products of g初乃8，功c浸（gη2乃4ノ，ψC（wαoC），吻D
（woαD〃∼かルノ），吻E（gη7乃C），ψF（wαα万）and吻G（wααG）were mapped into the
lipopolysaccharide（LPS）biosynthesis pathway［Raina and Georgopoulos，1991；Klein
et al．，2009；Kneidinger et al．，2002；Roncero and Casadaban，1992］． The products of
these genes are involved in synthesis of the heptose ur直t of
ADP−L−glycero−D−manno−heptose丘om sedoheptulose−7phosphate or encode early
heptosyl transfbrases fbr KDO−lipid A（ψC andψ万）and to fUrther extend the inner
core of LPS with glcosyltransfbrase（吻G）．ア6た」乙，露」乙（ろα加」B），アηゐE，ηψ1 andア040
encode peptidoglycan−associated outer membrane lipoproteins， and the products ofρα1，
’01ρand’01R are components fbr a complex structure fb㎜ing a biopolymer transpo丘er
［Gerding et al．，2007；K㎜pfbnkel and Braun，1993］．アc’M encodes a protein possibly
required fbr integriW of the outer membrane［Niba ETet al．，2007］． The the㎜o sensitive
phenotype caused by disrupted mutants of all of these genes was significantly
suppressed by the addition of Mg2＋ （Table 2．2， Figure S4）． Since Mg2＋is㎞o㎜to
stabilize the outer membrane structure by binding extracellularly［Nikaido，2003］， it i s
ass㎜ed that YdcL， Y塊L， YnbE， NlpI， YcdO， Pa1， TolQ， TolR and YciM act as
components or scaffbld proteins of the membrane to maintain outer membrane integrity，
especially at a high temperature． Similarly， our data suggest that Mg2＋is able to
stabilize the outer membrane stmcture when the LPS biosynthesis pathway becomes
defbctive．
Group C consists of∂加g，乃01C，1ワ7孟4，7協4 and 7πvC fbr DNA double−strand
break repair（DsBR）［Mot㎜edi et al．，1999］． DnaQ and Holc are epsilon and chi
subunits， respectively， of DNA polymerase IH［F月alkowska et al．，1997；39 Xiao et al．，
1993］，which is required fbr homologous recombination in DSBR［Adalns and West，
1996］．RuvA and RuvC act as DNA helicase and endonuclease， respectively［Adams
εmd West，1996；40 Bennett and West，1996］， befbre the replication restart in the DSBR
process， and PriA fUnctions as DNA helicase af㌃er the replication restart［Al−Deib et a1．，
1996］．The requirement of DSBR fbr survival at CHT suggests that DNA molecules are
su切ected more to double−strand breaks at a higher temperature． Interestingly， mutants
of all members in this group exhibited sensitivity to oxidative stress at 30°C． Therefbre，
it is thought that there is a strong connection between oxidative stress and DNA
doubIe−strand breaks．
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Group D includes genes fbr tRNA modification． Products of∫303，地θ、乙伽5B），
ア乃θM伽5（⊃，ア乃θ2＞伽3D）and y乃んP（’π＆4）have been defnonstrated to compose the
sulfhr−relay system［R（ヵas and Vasquez，2005；Ikeuchi et aL，2006；Dahl et al．，2008］．
IscS is a widely distributed cysteine desulfUrase that catalyzes desulfUration of
L−cysteine by trans色r of the su1㎞to its active−site cysteine to fb㎜apersu1且de group
（−SSH）， being responsible together with YheL， YheM， YheN and YhhP fbr biosynthesis
ofthe 2−thio modi且cation of 5−methy1㎜inomethyl−2−t㎞o肛idine（㎜5 s2U）［Reuchi et
al．，2006］and five diffbrent thio modifications in bacterial tRNAs［Lauhon，2002］． IscS
also works as a general sulfUr donor in various metabolic pathways［Mihara and Esaki，
2002］including biosynthesis of iron−sulfUr（Fe−S）cluster［Frazzon and Dean，2003］，
thiamine［Taylor et al．，1998］， nicotinic acid and branched−chain amino acids［Lauhon
and K㎜bampati，2000］． Additionally，1η∫α浸，〃η2σandか勧4 in ths group are involved
in tRNA modification． The mutations of genes related to sulfbr modification cause the
phenotype of sensitivity to anti−oxidation stress［Dahl et al．，2008］． Consistently， our
study provided evidence that mutants of this group exhibited hypersensitivity to
oxidative stress． YheL， YheM， YheN and YhhP， which mainly fUnction in t−RNA
modi丘cation［Lauhon，2002］， are conserved in the㎜otolerant bacteria in mesophiles
（see Table 2．3）， whereas∫3cε， a general sulfUr donor， is widely conserved in mesophiles．
These findings suggest that tRNA modifications presented here are indispensable fbr
growth at CHT．
Table 2．3 Distribution ofthe㎜otolerant genes in group D in various bacteria．
Bacteriaa
菰5・08 ア乃ε五 ア加ルf ン乃ε2＞ ア乃雇） 1蛎α匁 か㍑σ か麗オ
E30舵ガ0玩αCO〃
0
0
0
0
0
0
0
0
SαZ〃20ηθ1ぬθ〃彪γ∫oα
0
0
0
0
0
0
0
0
y診z3加∫α．ρθ5〃3
0
0
0
0
0
0
0
0
働∫9ε1zαプ7εκηθア’
0
0
0
0
0
0
0
0
κZεゐ3ゴθ1地Pηθπ〃20η∫αε
0
0
0
0
0
0
0
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aBacteria sho㎜here are representatives of species of which genomic sequences are available in databases．
“
o”and“x”represent the presence and absence ofthemotorelant genes in group D， respectivel》へ
Group E genes encode chaperones and a protease and thus contribute to the
cellular process of regulating heat shock response：伽αK and伽α1 encode a chaperone
and co−chaperone， respectively， fbr maturation of protein fblding or refblding of
unfolded proteins［Tomoyasu et al．，1998］， and漉gP encodes a chaperone／serine
protease located in the periplasm［Lipinska et al．，1990］． The indispensability of these
genes at CHT suggests that DnaK／DnaJ play a crucial role in dealing with unfblded
proteins caused by CHT and that DegP plays an important role in the removal of
damaged proteins that have accumulated at such a temperature．73θオin this group
encodes an anti−sigma飴ctor to keep sigma 24 inactive㎜der non−stress conditions． The
the㎜osensitivity caused by 75εオdisrupted mutation suggests that且ne tuning of the
intracellular level of active sigma 24 that regulates expression of chaperone or protease
一
21一
genes is somehow crucial fbr adaptation to the CHT condition． Alternatively， the
defbctive mutant of r5θオincreased sigma 24 activity， which in tum decreased the
production of outer membrane proteins via MicA or RybB as a sigma 24 regulon gene
［Valentin−Hansen et al．， 2007］， resulting in membrane 皿stability and
the㎜osensitiveness at CHT．
Genes in group F l）elong to the translation control apparatus． S6 encoded by
πρ3F interacts with the central domain of 16S rRNA and has been demonstrated to play
aregulatory rather than a structural role in the ribosome［Britton and Lupski et al．，
1997］．L36 encoded byη吻J is a component of the 50S sub㎜it of the ribosome， and its
disruption decreases the expression of 3εd7［Ikegami et al．，2005］， which encodes a
protein−conducting channel in the cytoplasmic membrane． DksA encoded by 4盈オ
fUnctions as a negative regulator fbr rRNA genes［Perron et al．，2005］． Overexpression
of DksA has been sho㎜to be a suppressor fbr a伽αK deletion mutation［Kang and
Craig，1990］and ensures replication completion by removing transcription roadblocks
［Tehranchi，2010］． SmpB encoded by遡ρβis a component of the trans−translation
process and per鉛㎜s rescue of stalled ribosomes with its binding pa丘ner，
trans飴r−messenger RNA［Watts et a1．，2009］． These lines of evidence suggest that
several constituents in translation pathways are crucial fbr survival at CHT．
Finally， genes in group G are related to cell division． AκθγC−encoded protein is
asite−specific recombinase［Bloor and Cranenburgh，2006］and is essential fbr
conversion of chromosome dimers to monomers during cell division．εηvC encodes a
component of the cell division machinery that is a direct regulator of the cell wall
hydrolase responsible fbr cell separation that is required fbr cell division［Uehara et al．，
2010］．DedD encoded by 4θ4D is a membrane−anchored periplasmic protein involved in
septation［Gerding et a1．，2009］and has been sho㎜to p韻icip翫e in c外okinesis
［Arends et al．，2010］．
The fUnctions of the remair亘ng genes，アbg瓦アo∫ハ4 and）ノ乃ん砿are un㎞o㎜．
Notably， the the㎜osensitiveness of their mutations was pa丘ially suppressed by the
addition of Mg2＋． It is thus likely that their gene products are related to cellular activities
similar to those in group B， C or G．
2．3．4Possible acαuisition of some thermotolerant genes by horセont劉1 gene transfbr
Two groups fbr outer membrane integrity and tRNA modification are almost
completely conserved in limited bacterial species with optimal growth at a relatively
high temperature（Table 2．4）． Of these group members， genes fbr the LPS biosynthesis
一
22一
pathway， some lipoproteins and the sulfUr−relay system are distributed in very limited
bacterial species including E雇θ70ゐo（フ’θ7∫αcεαθ（Tables 2．5 and 2．3）． The sulfUr−relay
system classified in tRNA modification has been demonstrated to modifンafbw
nucleotides of tRNA molecules， contributing to stabilization of their structule， and to be
required fbr survival at an extremely high temperature in 7乃θア〃20〃5’乃θrη2qワ乃∫1〃3［Shigi
et al・，2008］and it is also conserved in 7乃θr〃20αηαθ70Z）αc∫θ7’εηgooη9θη5’5「（Table♀・3）・
The mature LPS biosynthesis pathway fbr assembly of the outer membrane consists of
many enzyme reactions， which was fbund to be dispensable at a lower temperature．
Interestingly， this pathway is mostly conserved in 7乃θ朋o∂23ψbvめ吻アθ110w∫’oηガand
肪ε7〃20rηαθ70v必rわαc∫dα〃2∫ηovorαη3（Tal）le 2．5）． Enzymes in the LPS biosynthesis and
sulfUr−relay system in E． co1∫share about 40％sequence identity and about 50％
sequence similari呼to the coπesponding enzymes in the㎜ophilic bacteria． E． co1∫and
its closely related bacteria would thus have acquired these genes of the two groups
pres㎜ably by horizontal gene trans飴r du血g their evolution． Since the other丘ve
groups are widely cons6rved not only in the㎜otolerant mesophilic bacteria but also in
other mesophilic bacteria， they would be intrinsically present in E． co1二This is
consistent with the conserved natule of essentiality of the lipid A part of LPS and
essentiality of synthesis of lipid IVA but dispensability of enzymes involved in
extension of Kdo2−lipid A by various glycosyltransfbrases． This draws support ffom Re
（吻C）mutants with only tetraacylated lipid A exhibiting a very nalrow growth range
with ability to grow or旺y under slow growth conditions on mir直mal medi㎜aro㎜d
23°C［KIein et a1．，2009］， suggesting overall importance of outer membrane integrity at
CHT．
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3．3．5Expressional change caused by heat shock at C］HT
None of the the㎜otolerant genes identi丘ed in this study were fb㎜d to encode
HSPs previously identified in E． co1∫except fbr 4ηαろ伽α1く 漉91）and 4η09． To
examine whether the the㎜otolerant genes were up−regulated at CHT or not， we
examined transient change in expression of the genomic genes at CHT by DNA chip
analysis． The results showed that 42 genes and 111 genes were significantly
up−regulated and do㎜一regulated， respectively（Table 2．6）． The up−regulated genes were
classified mainly into genes involved in the cellular process， and the down−regulated
genes were classified into genes involved in energy metal）olism， transport／binding
protein and translation． However， none of the the㎜otolerant genes including 4θgP and
4加gas a heat−shock gene were identified as up−regulated genes except fbr伽αJ and
4η01（．Taken together with data shown above， it is possible that the chaperone systems
except飴r Dn訂／DnaK and GroEL／GroES are not neccesarily involved in the㎜otolerant
mechanisms acquired at CHT． Therefbre， it is likely that most products of
the㎜otolerant genes are not HSPs and that the organism possesses a speci且c set of
genes required fbr survival at CHT．
It is possible that some of the essential genes are cmcial fbr growth at CHT．
Such genes， however， could not be examined in this analysis because no disrupted
mutants fbr these genes are available other than the conditional mutants． We thus listed
essential genes with significant fluctuation in expression at CHT（Table 2．7）． gアo」砿
（g70」乙）encoding HSP was up−regulated， indicating the possibility that the gene product
contributes to survival at CHT． Consistently， it was reported that GroEL appears as a
mediator of evolution of extremely heat−resistant E． oo1∫cells［Rudolph et a1．，2010］． On
the other hand，90％of the down−regulated genes were mapped into the translation
pathway（Figure S 5）， encoding fbr components of ribosomal proteins． It is thus possible
that do㎜一regulation of ribosomal genes is one of the strategies fbr s㎜ival at CHT in
E．oo1ノ． Noteworthily， Alixθ’α∠reported that ribosome biogenesis in E． co1∫is high
temperature−sensitive and DnaK−dependant and predicted that high temperature causes a
severe limitation in DnaK／DnaJ to hamper ribosome assem『bly because heat−induced
misfblded proteins would titrate out all the f士ee DnaK／DnaJ［Al Refaii and Alix，2009；
Ren6 and Alix，2010］．
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Table 2．7 Essential genes significantly up−regulated and down−regulated at CHT．
Pathwaya
Gene
Cpn60 chaperonine
RNA degradation
9アo」班
tRNA−Leu
Transfbr RNA
1θ〃σ
Classificationa
Up−regulated
Down−regul ated
カゐG
Lipid metabolism
Fatty acid biosynthesis
Transcription
RNA polymerase
ηフo遵
Translation
16S rRNA processing protein
r∫1η
TransIation
Translation factors
ル誼
Translation
Ribosome
ηフ5P，ηワ19，ηフ5D，ηフ5κ
刑ρ5ハか脚Cηワ1P，ηワ5C，
アP1π耽ρ3S刑ρ1B，刑ρ1砿
ηワ1D，刑ρ1C，ηフ＆兀ψZJl
ηつ琵，ηフ5R
aClassification and Pathway accoding to the KEGG PATHWAY are sho㎜．
（http：／／www．genome．j p／keg9−bin／show＿organisrロ？menu＿type＝genome＿infb＆org＝eの．
2．3．6hrther consider紐tion on mechanisms fbr survival at CHT
Two groups of DNA double−strand repair and chaperone／proteinase genes may
contribute to endurance against oxidative stress in addition to CHT． Evidence that a
higher temperature results in acc㎜ulation of more oxidative stress［Noor et al．，2009］
and the finding that mutants of all members in both groups exhibited sensitivity to
oxidative stress allow us to speculate that oxidative stress is a main cause of DNA
double−strand breaks and of damage to proteins at CHT． Interestingly， oxidative stress is
involved in heat−induced cell death in 8． cε7θv’5∫oθ［Davidson et al．，1996］， which is
supported by the findings that overexpression of catalase and superoxide dismutase
genes could increase the degree of the㎜otolerance and that the the㎜otolerance is
一
34一
increased㎜der anaerobic conditions． We thus ass㎜e that CHT somehow causes
intracellular oxidative stress to elicit hannfUl effbcts on cells as a secondary stress．
Signi且cant suppression of the the㎜osensitive phenotype by a de飴ct in the
group of energy metabolism（Group A）by the addition of glucose suggests the
limitation of energy level at CHT in the organism． The limitation seems to be resolved
by altemative pathways that may generate ATP by glucose assimilation． The
requirement of ATP at CHT may be bonsistent with expression of ribosomal genes．
Many genes fbr ribosomal proteins were fb㎜d to be do㎜一regulated by exposure to
CHT， and the disrupted mutant of傭匁that encodes a negative regulator fbr rRNA
genes became the㎜o sensitive to CHT． These丘ndings and evidence that translation as a
ribosomal activi馴tilizes much ener部， up to about 90％of ener剖cons㎜ed in cells
［Le㎞inger and Cov，1993］， suggest that cells manage to reduce ener留cons㎜ption
under a severe conditiQn at CHT． Such saved energy would be utilized fbr other crucial
activities such as repair or degradation of damaged DNA or protein molecules． A
smooth translational process at CHT might also save energy， fbr which S6 and L360f
ribosomal proteins in addition to SmpB may have important fUnctions．
Several strategies fbr E． co1∫to s即ive at CHT were discovered． Most of them
may also be responsible fbr other stresses and are conserved even in mesophilic
bacteria． Early glycosyltransfbrases fbr LP S core biosynthesis fbr proper outer
membrane assembly an4 pe㎜eability baπier fUnction and the sulf腱一relay system fbr
tRNA modi丘cation might have been acquired鉛r the organism to per鉛㎜amain task
to survive at CHT． Considering the genetic conversion of non−the㎜otolerant to
the㎜otolerant bacteria， the two strategies might be applicable．
SupPrement lnfbrmation
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Figure Sl Growth of thermosensitive mumnts in LB匿iαuid culture飢dif距rent
tempemtures． Each 51 thermosensitive mutant straill（opened symbols）and the parental
strain， B W25113（closed symbols）， were grown in 30 ml LB medium at 37°C（c丘cles），45
°C（squares）， or 46°C（triangles）． At the t㎞es illdicated， turbidity at OD600 was measured．
A，group A；B， group B；C， group C；D， group D；E， group E；F， group F；qgroup G；H，
others．
一
39一
αcθE
αcεF
ρゴん1∼ αcεE αoεF 脚
［：＝：〉 ［：：＝：：⊃〔：＝：〉
αoεE αoεF えρ4
［＝：＝：：：：：：：）−E＝＝〉
ゆC η匂F り劔） 乃々rL
吻D
〈＝＝＝コ〈＝：：＝＝14■■■■■［：：⊃
ψF
吻C 吻F ψD
く＝＝＝コq■■■■■〈＝＝＝：：］
露L
dθr 脆五 塊M
〈＝：＝：：コq■■■■〈：＝＝コ
’019
y∂gC ω’9 ご01R
［：：：＞1■■膨［：：）
乃01C
vα1∫ 乃01C p召餌
く：＝＝：：＝＝コ＜■■〈：＝＝コ
ア乃θルfア海θLηワ5
ア乃εム
E＝）■■レ［＝〉
アたε1VアhεMア乃ε五
ア乃θルr
［＝）■■9・D
ノ乃80 ア乃θjvアぬθルrア乃ε乙
ア舵八F
［：：：〉一〔＝＞D
4ηαK
〈＝＝］ ［＝＝＝：＞
アαα∫ 4ηακ 4ηω「
3θo｝7ηワ〃ガηワ3Hηワ∫K
耽ρηが
［：：：）b［：＞E：〉
麗 ηつ3Fρr∫βηフ∫R
胆5F
〈＝コ ■」［⊃堤＝〉
Figure S2 Gene organizations around genes having either an essenti劉l gene or a
the㎜otoler劉nt gene as a j ust downstre劉m gene． Gene org㎝izations around 13
thermotolerant genes that have either an essential gene or a thermotolerant gene as a just
downstream gene are depicted． Black boxes represent identified 13thermotolerant genes．
Grey boxes represent essential or thermotolerant genes． The direction ofboxes shows the
dhection oftranscription．
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（B）
（A）
Gene
Strain
Gene
αc8F
BW25113
o‘8F
Str血n
BW25113 00εE：：肋η
ααぎ乏「：：㎞舷
Cyde 15 20 2530 15 2025 30
Cyde 15 20 25 30 15 2025 30
Gene＿＿＿＿＿＿竺＿＿＿＿＿＿
G・爬＿＿＿＿＿竺＿＿＿＿
Strain BW25113 αcεF：：加雌
ασθF：：肱P1
Strain BW25113
Cyde 15 20 2530 15 202530
■■■■■畷饗■■■■■配■
Cyde 15 20 2530 15 2025 30
■■■圖冨＝：■■囮昌昌iヨ
G￠ne rルF
G㎝e＿＿＿＿＿＿厘＿＿＿＿＿＿
Stram BW25113
Strain BW25U3 幽」
Cyde 15 20253Q 15 202530
Cyde 15 20 2530
Gene＿＿＿＿＿匹＿＿＿＿＿＿
鵬ne
Strain BW25H3 幽三＿
BW25113 幽」
■■唱：：■■■＝：
15 2025 30
ψc
Strain
Cyde 15 20 25 30 15 2025 30
Cyde l5 20 2530 15 2025 30
■■：＝■■＝＝
−
Gene ゴεr
Gene 4θr
Strain BW25113 幽乙＿
Str加n BW25】13 一幽一
Cycle 15 20 25 30 15 2025 30
Cyde 15 202530 15202530
■■■■国■■■■■
■■圏＝＝■■■囮
Gene ’01R
Gene ∫o’R
Strain BW25113 幽乙＿
Strain BW25113 』迦一
Cyde l5 202530 15 20253Q
■■圏■目■■＝＝＝：
Cyde l5 20 2530 15 202530
■■■＝＝＝■國：＝：：：
Gene vα’∫
Gene vα！∫
S廿ain BW25113 ho’『勉η
Strain BW25113
Cycle l5 20 2530 15 2025 30
ho！C：：加π
■■■巴巳■■一
Cycle 15 20 2530 15 202530
■■■＝：≡圏■■■＝＝：■
Gene＿＿＿＿＿＿匹＿＿＿＿＿＿
G。爬＿＿＿＿＿響と＿＿＿＿＿
StraiロBW25113 一幽＿
Str謡n BW25113
Cyde 15 202530 15 202530
Cyde 15 202530 15 202530
■匿＝：ヨ■圏＝冨「
Gene ．ソんεム
Stran BW25113
Cyde 15 202530
（詑ne
yんθL
Str由n BW25113 』幽」
15 2025 30
Cyde l5 20 25 30 15 2025 30
■■■■■唱題■■■国■
G㎝e＿＿＿＿＿些＿＿＿＿＿
G㎝。＿＿＿＿＿＿虹＿＿
S画n＿旦遡⊇＿ 一幽＿
Strain BW25113 幽」
Cycle l5 20 2530 15 202530
Cyd￠15 202530 15 202530
■■：＝：＝■国＝＝＝
■■■■＝ ■■■冨：：
Gene 4παノ
G￠ne 伽αノ
Strain BW25113 ゴ舩κ：舷艀
Str由n BW25113
Cyde l5 202530 15 202530
■■■圏：＝：■■■国團：コ
Cyde 15 20 2530 15 2025 30
■■■■国囮＝：■■：＝＝日
Gene＿＿＿＿＿＿皿＿＿＿＿＿
G㎝e＿＿＿＿＿＿鯉＿＿＿＿＿＿
S画nBW25113
Strain BVゾ25113
Cydc I5 20 253Q 15 2025 30
Cyde 15 20 2530
dπ口κ：：舷π
15 202530
Gene＿＿＿＿＿囲＿＿＿＿＿＿
Gene＿＿＿＿＿＿逆＿＿＿＿＿＿
Sぼain BW25113 幽一
Str組n BW25113 幽」
Cyde 15 202530 15202530
■圏＝コ5■■■■目
Cyde 15 20 2530 15 202530
■匿＝記5Eヨ■■■■圏昌
（D）
（C）
ダ懸轡露ノ霧ノだ
霧だ壽嫁ノノノノだ
Figure S3 Testing of possible polaref笛ects by the￠ρ乃insertion． Tbtal RNA was prepared
廿om cells￠u壮ured at 37°C（A， C）and 47°C（B， D）as described in Materials and methods．
RTPCR was perfbrmed with pr㎞ers specific fbr a just downstream gene ofeach
thermotolerant gene to amp lify about 500−bp DNA廿agments．（A and B）After RT reaction，
PCR was perfb㎜ed 15，20，25 and 30 cycles and the produ㏄s were analyzed．（C and D）As
acontrol， each total RNA（10μg）was submitted to l．2％agaro se gel electrophoresis and
staining with ethidium bromide．
一
41一
C
B
A
（A）
D
E
F
G H
0．6
ぎ
05
0．4
03
隔0％GlロOD5c
0．ユ
o．l
ロ0．5％Gluoo鵠
o
∼彦柵騰鯉ぎ鞭鍵3ミ鱗綿ξ鞍錨翻欝ぎ躍睾辮ざ
（B）
むめ
誤・5
0 〔レ．4
0」
02
■OmM MgCl2
0．1
0
鳶終騨響舞ぎβ鉾ぎざ運書2ミ鐙撃誕錠鞭誉慧膏3鞭匿ぎ含謬繹紳謹
口20mMM巳q2
ぎ
（C）
しう
鴫：：1曲1曲曲L曲曲1，【1山1山翫旺h』II曲m呂蹴奴
y齢蝉轄騨構醗季轡欝麟緯麟聯♂欝岬躍
Figure S4 E餓｝cts of addltion of glucose and MgC12 and sensitivity to H202．
Thermosensitive mutant strains are shown by gene names． Growth conditions are described
in Materials and methods． B lack and white columns represent turbidity under the oond辻ions
with or without supplements（0．5％glucose（A）or 20 mM MgCl2（B））or O．5 mM H202（C）．
！！L3
鰻L4
耽ρ∬ 刑ρ1C
ηフπ）
聾SlO
1！L36
！！S13
！！Sll
鰻L23
！！L2
ワ1〃「 耽μB
11S19
ηフ53
！lS3
1！L22
耽ρ17 ψ∫C
1！S4
弓ρ5M 弓ρ31（ 弓ρ5D
鯉Ll7
！！L13
！！S9
耽ρ19
L7／L12
胆
L9
LlO
L1
Ll1
ψ
1！Sl8
盟S6
ηフ3R
Sl6
聾L19
κρ5P
Figure S5 Down−regu且ated genes fbr ribosoma亘proteins． Systematic analysis ofgene
且mction was perfbrmed with a database ofKEGG PArHWAY Down−regulated genes fbr
ribosomal proteins were mapped into 60perons．
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匝亟］［亟コ
ψ1P
ηフ溺C
Chapter 3
Molecular metabolism of thermotolemnce in eth劉nologenic
themotolemnt 2ン〃20〃20πα5〃ωゐ〃畝5紐PPIicable fbr high temper舳re
艶rmenmtion
Astract
To identifンthermotolerant genes responsible fbr growth of the ethanologenic
the㎜otolerant Z脚ゐ∫118 at critical high temperature（CHT）， TISTR 548 strain that was
chosen as a the㎜otolerant strain was su切ected to transposon mutagenesis via
transcor噸ugation with a mobilizable plasmid harboring the transposable element（Tn10）
and growth experiment at 39°C， CHT fbr the strain． Among about 4，0000f the
transcor亘ugants obtained，42 mutants that were fbund to be dr㎜atically defbctive in
growth翫the CHT， which were selected as the㎜osensitive mutant strains． The
inse丘ion site of Tn10 within the genome was then dete㎜ined by the㎜al asy㎜etric
interlaced−（TAIL）PCR fbllowed by DNA sequencing． As a result，17 genes related to
the the㎜otolerance have been identi且ed． Some of these were related to membrane
biosynthesis and lipid metabolism， recombination fbr DNA repair and replication，
tRNA modification， transportation system， which may have a direct or in direct relation
to the the㎜otolerance mechanism． Interestingly， the results also revealed a partial
overlapping between genes required fbr the themotolerance and those fbr tolerance to
other stresses． Our findings provide molecular mechanisms㎜derlying a survival of Z
脚oわ∫1∫5at CHT which may advantages in production of ethanol and other usefUl
materials at hi gh temperature．
3．11ntroduction
Z〃20ろ∫1’3as a Gram−negative， non−spore fbmling and polar flagellated
bacteri㎜has been isolated丘om sug飢cane as well as alcoholic beverages such as
A丘ican palm wine and is㎞o㎜to cause cider sic㎞ess and spoiling of beer［Swings
and De Ley，1977］． Gibbs and DeMoss［1954］discovered its anaerobic catabolism of
glucose fbllowing the discovery of the Entner−Doudoroff（ED）pathway in the early
1950s． The ED．pathway utilizes l mol ofglucose to yield 2 mol ofpyruvate， which are
一
43一
then decarboxylated to acetaldehyde and reduced to ethanol， which was lower than
that of the Emden−Meyerhof pathway of the conventional ethanol producer yeast
［Panesar et a1．，2006］and is generally recognized as safb（GRAS）status［Yang et al．，
2010］．The world wide interest have thus fbcused on the application of this organism
as an ef丘cient microorganism fbr production of the bio−fUel［Osman and Ingrarn，
1987；Ingram et al．，1998；Altintas et al．，2006；Cazetta et al．，2007；Bai et a1．，2008；
Agrawal et al．，2011］and of usefUI materials such as oligosaccharide；lactosucrose
［Han et al．，2009］and levan［Chiang et al．，2009］that can be apPlied fbr fbod and
pha㎜aceutical industries［Calazans et al．，1997］and medicine［Yoo et al．，2004］．
Z加oわ11∫5TISTR 548 is one of the㎜otolerant strains［Sootsuwan et al．，2008］
isolated丘om Thailand， and can grow at 39°C［Charoensuk et al．，2011］， which is about
10°Cover the reported optimum temperature of Z脚ろ∫1∫5［Swing and De Ley，19771．
The definition of thermotolerance is that a mesophile is able to grow at about 10°C to
15°Chigher than the general sarne species［Saeki et al．，1997］．
There are a number of advantages in applic凱ion of the㎜ophilic microorganisms
fbr production of ethanol and usefUl materials：reduction in cooling cost， higher
saccharification and fbrmentation rates， continuous ethanol removal and reduced
contamination［Singh et al．，1998］． However， in even the㎜otolerant strains， heat stress
may impact on their growth viability［Basso et a1．，2008；Babiker et al．，2010；］and also
they may cause stuck角㎜entations， especially when other琵㎜entation魚ctors reach
critical values， i．e． low pH， high ethanol content， high osmolarity， nutrient supply and
temperature etc．［Piper，1995；Ca㎜elo，1998；Ciani，2006；Pizarro，2007；Coleman et
al．，2007；Gibson et al．，2007］．
When exposed to enviro㎜ental s甘ess， cells alter their transcriptional progr㎜，
resulting in the i㎜ediate do㎜一regulation of層housekeeping genes and dramatic
increase of expression of a set of crucial defbnse and／or adaptation genes which are
essential fbr cell viability［Ruiz−Roig et al．，2010］， and under more sever situation，
protein denaturation， transient cell cycle arrest and variations in membrane fluidity and
structure occur［Benschoter and Ingram，1986］． Cells can adapt to relatively mild（not
sever）stress， which is㎞own as the heat stress response［1対ezman，2004；van der Veen
et al．，2007］to protect their components［Guyot et al．，2005］． However， mechanisms
underlying heat tolerance may be complicated in which many diffbrent genes are
ass㎜ed to be involved but the exact mechanism has not been血lly delineated［Je岱ies
and Jin，2000］．
Since the ethano1色㎜entation process is exothe㎜ic［Uden，1981；Ghose，2004］，
ethanologenic microorganisms seem to be exposed to heat stress in addition to other
一
44一
stresses including ethanol［Attfield，1997；Wei et al．，2007］． The effbct of heat and
ethanol stresses on the protein expression pattem in Z加oゐ’1∫5 is evident in several
polypeptides that associated with the cell envelope ffaction in addition to specific heat
shock proteins［Michel and Starka，1986；Thanonkeo 2007］． Some of them would be
involved in heat and ethanol tolerance mechanisms in the organism Only limited
i㎡b㎜翫ion is available on the capacity of the the㎜otolerant Z吻o励5 to grow at high
temperatures． The molecular mechanisms that allow Z〃20ろ’1∫5 cells to survive at high
tempera血e enviro㎜ents are very use釦1飴r high−tempera傭e飴㎜entation of
bioethanol and other usefUl materials．
This study aims to identi鯨the the㎜otolerant mechanism suppo貢ing the growth
of themlotolerant Z〃20わ’1ゴ3 at high temperature． Comparison of two TISTR strains in
Thailand， ZM4， NCIMB 11163 and LMG revealed that TISTR 548 was the most
the㎜otolerant． TISTR 548 was then su切ected to transposon mutagenesis， via
transcor巾gation with a mobilizable plasmid harboring the transposable element（Tn10）
and the㎜osensitive mutants were isolated． The mutation site of each mutant was
dete㎜ined by the㎜al asy㎜etdc interlaced−（TAIL）PCR鉛llowed by DNA
sequencing and the㎜otolerant genes were identi且ed． These genes were then
categorized and compared with those in、E oo1∫shown in the 2nd Chapter．
3．2Materials and methods
3．2．l Materials
ADNA sequencing Kit（ABI PRJSM⑪Te㎜inator v 3．1 Cycle sequencing
Kit）was obtained丘om Applied Biosystem Japan． Oligonucleotide primers were
synthesized by Proligo Japan K．K．（Tokyo， Japan）． Other chemicals were all of
㎝al外ical grade and obtained丘om co㎜ercial so砿ces．
3．2．2Microorganisms
Z励1115strains used in this study were grown in YPD medi㎜consisting of 3
gof glucos6，0．5 g of peptone and O．3 g of yeast extract in 100 ml of IEW．ρells
cultured fbr g h and 24 h， these te㎜s conespond to optical densities of about O．8 and
2．5at 550㎜㎜der a sha㎞ng condition and about 1．5 and 3．O at 550㎜㎜der a static
condition， respectively， were used as exponential−and stationary−phase samples，
respectively． E． co1∫Sl7−1 carrying plasmids of the pSUP2021 Tn10was gro㎜in LB
一
45一
medium supplemented with 125 μg／ml of tetracycline and Z 〃20ゐ∫1’5
transposon−inserted mutants were grown in YPD medium containing 12．5μg／ml of
tetracyCline．
3．2．3 Growth劉nd eth紐nol production of Z〃霊oゐ漉5 strains
Z吻o励5TISTR 548 and 405 repo丘ed as the the㎜otolerant strain［Sootsuwan
et a1．，2008；Charoensuk et al．，2011］were compared in growth with the other type
strains of ZM4 strain［Seo et al．，2005］， NCIMB 11163［Kouvelis et al．，2009］and
LMG． Cells were grow on YP contai血g 3％glucose at 30°C fbr 18h， and then diluted
cells to ten times by YPD medium． Fiveμl of each serial diluted cell was spotted onto
an YPD agar plate with or without supplemented agents， and incubated at 30°C or 37°C．
Their growth was observed and their pictures were taken after 48 h incubation． Growth
and ethanol production in YPD medi㎜were caπied out in triplicate鉛r each strain．
3．2．4 Conjug統tion
．E． co1∫S17−1 calrying pSUP2021 Tn10 as a donor strain fbr corゆgal mating
was routinely gro㎜in LB medi㎜containing 125μ9／ml of tetracycline under l oQ
rpm−shaking condition at 37°C． The recipient Z〃70ろ∫1∫5 TISTR 548 was gro㎜in
YPD medium at 30°C㎜der a static condition． After both strains were grown to
mid−log phase， about O．80f OD600㎝d washed three times with LB broth medi㎜
and each bacteri㎜was recovered by centri釦瑛ion飴r l min翫5，000Φm then
mixed at a ratio of donor／recipient of 3：2， incubated fbr 3 h at 30°C to allow their
mating and fUrther incubated at 30°C fbr 5 h on the surf亘ce of LB agar plates with 15
μ1dot spot of suspension of the mixed donor and recipient cells． After mating， the
cells were recovered and re−suspended in YPD medi㎜， and then spread on YPD
agar plates containing O．2％of acetic acid and 12．5μg／ml tetracycline HCl．
Tn10−inserted mutants appeared after 3 days at 30°C， which were subsequently
screene母fbr fhrther experiments．
3．2．5 Screening of thermosensitive mutant strains
The Hrst screening was perfb㎜ed on YPD agar plates． Among about 4，000
transcor巾gants obtained were gro㎜at 30°C and 39°C， mutant strains that could not
grow or poorly grew on the plates were selected fbr the 2nd screening． The second
一
46一
screening was per飴㎜ed in l ml ofYPD medi㎜at 30°C and 39°C鉛r 24 h㎜der a
static condition with the initial inoculation at O．050f OD550． The mutant strains that
gave the OD5501ess than a half of that of the parent strain were selected fbr the final
screening， which was per飴㎜ed in 30 ml of YPD medi㎜㎜der the s㎜e condition of
the 2nd screer直ng． The mutant strains that gave the OD550 in the significant reduction
compared to that of the parent strain were selected and defined as themlosensitive
mutants．
3．2．6Effbct of various stresses on growth of thermosensitive mutants
To dete㎜ine the overlapping ef飴ct between high temperature and other stresses，
the㎜o sensitive mutants were s呵ected to osmotic， ethanol， oxidative and antibiotic
stress． Gro舳ability of the㎜osensitive mutants was dete㎜ined on YPD agar plates
supplemented with 12％（w／v）of glucose，2．5％（v／v）of ethanol or 2 mM H202．Cells
gro㎜in YPD medi㎜at 30°C飴r 18h under a static condition were sehally diluted
with YPD medi㎜， and 5μl of each diluted sample was spo賃ed on YPD ag訂plates
with or without supplemented agents and incubated at 30°C fbr 48 h． Growth ability test
was carried out in duplicate fbr each strain．
3．2．71）NA manipulation
Genomic DNA isolation and other general molecular biology techr丘ques were
perfbrmed as described by Sambrook et al［30Sambrook et al．，1989］． Concentration of
isolated genomic DNA was measured by using NanodroP（Nanodr6p Tec㎞010gies，
Wilmington， DE）．
3．2．8 1dentification of transposon （Tn10）−inserted site in the genome of
thermosensitive mutants by thermal紐symmetric interlaced（TAIL）−PCR and
nucleotide sequencing
Since transposon（Tn10）was randomly inserted into the genome in this
experiment， the u直㎞o㎜site inse式ed of Tn10was dete㎜ined by TAIL−PCR as
reported previously［Lui et a1．，1995］with several modifications as described below．
Three serials of PCR were perfb㎜ed on MycyclerTM the㎜al cycler（Bio−Rad）in
diffbrent conditions（Table 3．1）with specific primers， TnISR−1
一
47一
（GATCCTCTCGTTTGTTGCGGTCAGGCC）［Deeraksa et al．，2005］， TnISR−1．5
（AGGGCTGCTAAAGGAAGCGG）and TnISR
（ACGAAGCGCAAAGAGGAAGCAGG）together with degenerated primer， AD2
（GTNCGASWCANAWGTT）［Lui et al．，1995］． The first PCR was carried out in a 50
μlof mixture containing 10 ng of chromosomal DNA，5．0μM of specific primer
TnISR1，25μM of AD2 primer，500μM of each dNTP，0．5 U PrimSTAR（Takara）， a
buffbr supplied fbr enzyme and sterilized distilled water． The product of l st PCR was
50−times diluted and used as a template fbr the 2nd PCR in the same reaction mixture as
the lst PCR except that the TnISR1．5 was used as a specific primer． The 3「d PCR was
similarly per飴㎜ed as the 2nd PCR with a speci且c primer TnISR2 and 125μM ofAD2．
The products丘om 2nd or 3「d PCR were pロi丘ed by using a col㎜伽a）㎝d used as
atemplate fbr the reaction of nucleotide sequencing． The product of sequencing reaction
was then sulガected to sequence analysis using an ABI PRISM 310（PE Biosystems）．
Table 3．1 Reaction parameters of TAIL−PCR fbr amphfication ofthe丘ahking region of
Tn10 inse丘ed in the the㎜o semitive muta血strains of Z吻ooろノ1’3 TISTR 548 with the
mod置ed丘om previous report（Liu and Whitter，1995）．
Reaction Steps
Number of
Primer combinatio n
1st PCR
cycles
1
1
（TnlSR−1／AD2）
2
5
93°C，1m血；95°C，1min
94°C，30sec；64°C，1rn血；72°C，2min
3
1
94°C，30 sec；25°C，1 min；ramping to
4
15
72°C，3nユ血；then 72°C，2m辻L
94°C，30sec；64°C，1］［nin；72°C，2rnin；
Cycle（supercycle）parameter
94°C，30sec；64°C，1min；72°C，2mjh；
94 °C， 30 sec； 44°C， 1 1nin； 2 n血；
72°C，2mh1
2nd PCR
1
15
94°C，30sec；64°C，1rnin；72°C，2．5
（TnlSR−1．5／AD2）
min；94°C，30 sec；64°C，1rn血；72°C，
2．511血1；94°C，30sec；44°C，1 n1辻L；2
1r血1；72°C，2．51nin
3「dPCR
1
1
94°C，1rnin；
（TnISR−2／AD2）
2
30
94°C，30sec；50°C， 1min；72°C，2．5
血
一
48一
3．2．9Comparison of nucleotide sequences of flanking regions of Tn10 inserted with
those in database
Nucleotide sequences of flanking regions of Tn10 inserted in each
the㎜osensitive mutant were compared with those in the DDBJ database by using
BLAST［Altschul et al．，1990］． F顧her comp飢ison and alig㎜ent of nucleotide
sequences were conducted by using GENETYX（Software Development， Tokyo， Japan）．
The phylogenetic tree was constructed using CLASTAL W（DDBJ）．
3．2．10Analytical procedures
Ethanol㎝d glucose concentrations in the medi㎜were meas皿ed by hgh
perfb㎜ance liquid chromatography（HPLC）（Hitachi Chemical， Japan）using a Gelpack
GL−C610−S col㎜（7．8 x 300㎜）． A medi㎜丘action was prep飢ed as a supema伽t
after a low−speed centrifUgation of cultures at diffbrent cultivation time points and
diluted 10times befb士e applying to HPLC． Using HPLC with a reflective index detector，
chromatography was perfb㎜ed at 60°C and degassed water was used as a mobile phase
at a且ow rate of O．3 ml／min． Concentrations of ethanol包nd glucose were dete㎜ined by
comparing a detected area with that ofthe corresponding standard sample．
3．2．11Statistics
Unless otherwise indicated， mean and standard deviations（SD）were calculated
丘om data of at least three independent experiments． The variations l）etween the
experiments were estimated by SD， and the statistical significance of changes was
estimated by two−sample’−test assuming equal yariances．＊＊＊，＊＊and＊mean、ρ＜0．001，
＜0．01and＜0．05， respectively． The level of significance was accepted aψ≦0．05．
3．3 Results
3．3．1Comp紐rative growth and ethanol production of thermotolemnt Z翅oゐ’伽
with the other type strains
Zη20ゐ∫1∫5TISTR 548 and 405 as a the㎜otolerant strain［Sootsuwan et al．，
2008；Charoensuk et al．，2011］was compared in growth and ethanol production with
other the㎜osensitive type strains of ZM4［Seo et al．，2005］and NCIMB 11163
一
49一
［Kouvelis et al．，2009］at high temperature． A phylogenetic tree（Figure 3．1）with 16S
rDNA revealed that the two the㎜otolerant strains were closely related to the two
therrnosensitive strains．
The growth ability of cells was examined in YPD medi㎜containing 3％（w／v）
glucose with or without 2．5％v／v ethanol or 3 mM H202 and in YPD medium
containing 12％（w／v）glucose at high temperature after 48 h． As shown in Figure 3．2，
only Z溺oわ’1∫3 TISTR 548 and 405 strains could grow well at high temperature． By the
evaluation ofthe growth ability and the ethanol productivity at high temperature（Figure
3．3），TISTR 548 was cho sen fbr fUrther experiments．
ZM405a
NCIB 11163
63
LMG457
ZM4
ZM548a
ZM550b
〇、001
Fig．3．1 Phylogenic tree analysis of l 6 s rRNA ofthe thermotolerant Z魏oδ’1ご∫TISTR
548and other type strains．
（A）
（B）
37°C
30°C
＜ ZM4
＜ TISTR 548
＜ TIsm 405
＜LMG
＜NCDMB l l 163
（C） D
2．5％（v／v）Ethano1 3mM H202
一
＜ ZM4
＜ TISTR 548
＜TISTR 405
＜LMG
＜NCIMB ll163
Figure 3．2 Comparison ofgrowth abilities ofthe Zη20わ∫1∫εTISTR 548 with other type
stra㎞s under stresses condition．
Growth ability ofZ．ηωわ∫1∫∬かα燃on YPD agar plate；cells suspellsion of l 8 h with
serial dilution were spotted on the YPD containhlg 3％glucose agar plates and
㎞cubated at 30°C（A）and 37°C（B）， and on the YPD conta血ing 3％ghlcose agar plates
supplemented with 2．5％v／v ethanol（C）or 3mM H202（D）at 30°C． Photos shown are
representatlves・
一
50一
（B）
（A）
4．0
＿
十548一く）＿405−［｝−ZM4
4．0
△−548＿O＿405■｛｝−ZM4
3，0
3．0
鵠
り
需
ざ2・o
自
2．0
o
o
1．0
1．0
0．0
0．0
0
12 24 36
12
0
48
24 36
48
Time（h）
Time（h）
σ））
（C）
3．0
3．0
一
△＿548欄O＿405一口＿Z《へ4
ε
ε
ξ92．0
き 2．0
茗
宅
鎖 1．o
ヨ 1．o
毫
函
0．0
0．0
0
12 24 36
48
0
12 24 36
48
Time（h）
Time（h）
Fig．3．3 Growth of Z初oろ∫1∫3 strains in YPD medium under a static condition． Growth
ability was compared in 30 ml ofYPD containing 3％glucose under a static condition
at 30°C（A）and 39°C（B）， and at the same t㎞e ethano l concentration at 30°C（C）and
39°C（D）was measured． Reported values are the mean（±SD）of three independent
experiments，pvalues were calculated by two−sample’−test assuming equal variances．
3．3．2Screening of thermosensitive mutants and
identification of tbermotolerant
genes in Z〃20わ〃菰∫
To obtain the㎜osensitive mutants of Z濯o旙3 TISTR 548， a mobilizable
plasmid hearing the transposable element（Tn10）was transfbrred丘om E5c乃θ7／c痂αco1∫
S17．1 as a donor to Z吻oろ∫1’5 TISTR 548 as a recipient． About 4，000 transco切ugants
obtained were cultivated on YPD plates at 30°C or 39°C to observe a the㎜osensitive
phenotype such an absence of growth or smaller colonies at high temperatures． As the
result，126 mutant strains were selected as candidates of the㎜osensitive mutants． The
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growth phenotype of these mutants were fUrther examined by measuring cell growth at
39°C，and finally 42 mutants with a strong defbctive growth were obtained， which
exhibited significantly defbctive growth in YPD medium at 39°C but not at 30°C when
compared with the parental strain（Figure 3．4）．
（A）
1．5
§
≧
2 1．0
6幻
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〆♂・蹴麟詑謝・航評ぜ灘就♂
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0．0
Figure 3．4 Relative growths ofthe㎜osensitive mutant Z．配o旙3 strains with its
parental strahl． Cells were grown in YPD medium under static condition， The O．05
initial OD550 nm ofinoculums were used to grown cells in 30 ml ofYPD containing 3％
glucose under static condition． Growth at 30°C（A）and 39°C（B）， and ethanol
production at 30°C（C）and 39°C（D）． Reported values are the mean（±SD）ofthree
independent experiments．ρvalues were calculated by two−sample’−test assuming equal
varlances．
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3．3．31dentific飢ion of possible thermotolemnt genes
The inse質ion site of Tn10 in the genome of each the㎜osensitive mutant was
dete㎜ined by the㎜al asy㎜etric interlaced（TAIL）−PCR㎝d nucleotide sequencing．
This dete㎜ination revealed the genomic sequences flanking Tn10 inserted in the
genome（Table 3．2）． The nucleotide sequences were then compared with the genome
sequences of the type strain ZM4［Seo et al．，2005］and NCIMB 11163［Kouvelis et al．，
2009］．Genes disrupted by the inse質ion of Tn10 were ass㎜ed to be the㎜otolerant
genes if there was no polar effbct of Tn10insertion on the downstre㎜genes， and thus
42possible the㎜otolerant genes were identi且ed by the analysis． Of these，23 genes
were fb㎜d to be duplicated， and 19㎜ique genes were identi且ed． Interestingly， one
gene encoding transposase is absent ffom the type strain ZM4 genome． This is not
s即rising because there is the di脱rent n㎜ber of genes present in the same specie，
which allows each strain to exhibit individual characteristics［Welch et al．，2002；Seo et
al．，2005；Kouvelis et al．，2009］．
3．3．4Ef艶ct of other stresses on thermotolerant mu伽nts
In E． ooll， about 60％ofthe㎜otolerant genes have been sho㎜to be
oxidative−stress tolerant genes（see Chapter 2）． Effbcts of osmotic， ethanol and oxidative
stresses were thus examined on YPD plates． As expected， a pa丘of the the㎜osensitive
mutants also exhibited defbctive growth on other stresses（Table 3．2）． Therefbre， it is
ass㎜ed that there is some coπelation between high t6mpera傭e and other stresses in Z
吻oゐ∫1∫5as reported in a conventional yeast［Auesukaree et al．，2009］and E． oo1∫
［G㎜asekera et a1．，2008］．
3．41Discussion
The㎜otolerant Z吻o撚TISTR 548， which was fbmd to be an ef丘cient
ethanol producer at high temperature compared to other type strains， was su切ected to
transposon mutagenesis to obtain the㎜osensitive mutants at CHT． The analysis of
the㎜osensitive mutants led to identi取17possible the㎜otolerant genes． On the basis
of the evidence that Z〃ωゐ∫1∫5 possesses about 2，000 genes in its genomes［Seo et al．，
2005；Kouvelis et al．，2009］， the ratio of possible the㎜otolerant genes to total genomic
genes in Z〃20ゐノ1∫5 was about O．85％， which was nearly equivalent to that in E． co1∫（see
Chapter 2）． The possible the㎜otolerant genes were血nctionally categorized into 8
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groups as shown below． Notably， all groups categorized except fbr anti−oxidative stress
were fbund in E． co1∫， suggesting that a similar molecular mechanism fUnctions in
survival at CHT．
（i）Membrane stabilization：squalene hopene cyclase（ZMO 1548）encoded by
3乃cis involved in the biosynthesis of pentacycline trite甲anoid lipids，㎞own as a
hopanoids biosynthesis pathway． Hopanoids is the prokaryotic membrane component
［He㎜ahs et al．，1991］equivalent to sterols in eukaryotes and is thought to protect
bacterial cells by reducing molecular pemユeability and increasing membrane stability
丘om enviro㎜ental stresses， such as high temperature， hgh ethanol concentrations， or
㎞gh oxygen levels［Ourisson and Ro㎞er，1992；Be燗et al．，1993］． It has been thought
that the acquisition of hopanoids is an evolutionary adaptation fbr survival in the
presence of ethanol in Z 吻oろ∫1’5［Carey and Ingr㎜，1983］． ZMOO56 is
gluco samine／丘uctose−6−pho sphate aminotransfbrase（GFAT；EC 2．6．1．16）and catalyzes
the鉛㎜ation of glucos㎜ine−6−phosph翫e using glut㎜ine as㎝㎜o㎡a donor．
GFAT is the first and rate−limiting enzyme in the hexosamine biosynthetic pathway
（HBP）． This nucleotide sugar is essential fbr the R）rrnation of a plethora of
glycocor藪ugates fbr the peptidoglycan macromolecule in prokaryotes［Badet et al．，
1987］．The disrupti6n of GFAT gene is vital in some species， but these mutants can
res㎜e gro槻h when glucosamine is added to the media［Smiht et al．，1996；Bulik et al．，
2003；Authur et al．，2004］． The other is a membrane−span㎡ng Protein TolQ（zMoo161）
which is one component fbr a complex structure鉛㎜ing a biopolymer廿anspo茸er
［Kamp琵nkel and Bra㎜1993］． The mutation of this gene suf飴rs delayed outer
membrane（OM）invagination and contains large OM blebs at constriction sites and cell
poles［Gerding，2007］．
（ii）DNA repair：DNA repair protein RadC encoded byアodC（ZMOO663）
fUnctions specifically in recombination repair that is associated with the replication fbrk
and required fbr growth−medium−dependent repair of DNA strand breaks like 7θc浸that
required fbr virtually all recombination stimulated by伽o」B107［Saveson and Lovett，
1999］．During competence，γα4C is specifically co−induced with six’flanking genes，
御妖（アα♂C）一御7θ．BCD−〃z∫ηCD， encoding proteins involved in cell shape dete㎜ination
and septum placement． RadC thus could have a fUnction related to cell envelope
metabolism［Attaiech et al．，2008］． Exonuclease VII large subunit XseA encoded by
κ5θオ（ZMOO300）is implicated in the resection of the nicked mismatched strand during
bacterial methyl−directed mismatch repair pathway［Harris et al．，1998］that is used to
repair base mutations thereby keeping genome mutation丘equencies low［Larrea et al．，
2008］．Unexpected， all two mutant strains in this group were not sensitive to 2 mM
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H202． This may be explained by the飴ct that Z励1115 as a血cultative bacteri㎜must
have defbnse mechanisms against oxidative stress［Seo et al．，2005］and the severe
growth was observed under coupled stresses of 2 mM H202 with high temperature（data
not sho㎜）． Intracellular ROS that causes the DNA damage［Condon，1987；Zagorski，
2009］increases as temperature increases INoor et al．，2009］． Cells could maintain their
survival ability㎜der such conditions by DNA repair system．
（iii）tRNA modification：tRNA／rRNA meth）荏ransfbrase SpoU encoded by
ZMO1328 is involved in modification of tRNA molecules and may contribute to
stabilization of ribosome three−dimensional stmcture［Decatur and Fo㎜ier 2002］． A
number of additional modi且cations in RNAs provide advantages㎜der particular
conditions， such as con飴rring resistance against ribosome−targeting antibiotics［M㎜
et al．，2001；Toh et al．，2008］． However， the physiological roles of the vast m句ority of
modified nucleosides in rRNA remain un㎞own［Ero et al．，2008］． Hereby， I propose
that tRNA methyltransfbrase is indispensable fbr the growth of Z脚oゐノ1∫5 at CHT．
（iv）Chaperorゾprotease：metallopeptidase encoded by ZMO1422 contributes to
the cellular process of regulating heat shock response in the wide−range organisms
including−alian㎝d bacteria田u㎝g et a1．，2008］．
（v）Translation：ZMO 1897 is a sub㎜it of pre−protein translocate involved in
protein secretion to the periplasmic space or outer membrane．
（vi）Cell division：ZMOO236 is one of ATPases fUnctioning as a chromosome
partitioning−1ike protein and may be essential fbr conversion of chromosome dimer to
monomer during cell division［Leonard et al．，2005］．
（vii）Anti−o琴idative stress：xanthine−uracil pe㎜ease encoded by ZMOO969 is
involved in purine salvage and metabolism［Christiansen et a1．，1997］． Xanthine is one
of a subset of all bases of DNA damage generated by ROS［Kow，2002］． Oxidized
xanthine by xanthine oxidase was generated by periplasmic superoxide anions［Borsetti
et al．，2005］， that causes damage to membrane lipids， cellular proteins and DNA
molecules． In 42 the㎜osensitive mutants，21 xanthine−uracil pe㎜ease mutants were
obtained， which implied that this gene was the hot spot fbr insertion site fbr transposon
Tn10 in Z吻oゐ’1∫5． ZMO1965 encodes a predicted metal−binding protein． ZMOO935
encodes glutathione−S−transfbrase． This enzyme family is a superfamily of enzymes that
play a crucial role in cellular detoxi丘cation such as biotransfb㎜ation of toxic
compounds， protection against several stresses and antibacterial dmg resistance［La
Roche and Leisinger，1990；Masai et al．，1993；Favaloro et al．，2000；Orser et aL，1993］．
（viii）Un㎞own fαnction：noteworthy， about 25％of the identified genes encode
un㎞own proteins． TrpR−binging protein WrbA（ZMO1335）， DUF 162（ZMOO695），
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conserved hypothetical proteins（ZMOOO21）and（ZMO1243）， and hypothetical protein
（Z∂10＿0951）．Interestingly， WrbA fUnctions as an accessory element in blocking
TrpR−specific transcriptional process［Yang et al．，1993］of which expression increased
at the early stage of the stationary phase． It could play a role in preparing the ceIl fbr
long−te㎜maintenance under stress conditions［Chang，2002］． WrbA is also repo貢ed as
the multimeric flavodoxin−like proteins血mily［Grandori，1998］and designated as a
new type（type IV）of NAD（P）H：quinone oxidoreductases which protect cells against
oxidative stress［Patridge and Ferry，2006］mder stationary phase or at high temperature
［Soares et al．，2010；Noor et al．，2009］．
SurPrisingly， the latest protein is hypothetical protein（za10＿0951）， which is
absent in the genome of the type strain ZM4and shares a high similarity to transposase
（GOX2685）iM㈱ろαc’θr卿θ祝rlαη〃51FO3283［As㎜a et al．，2009］． Transposase is
one of DNA−binding enzymes and a member in the polynucleotidyl transfbrase
superfamily that catalyzes‘cut−and−paste’or‘copy−and−paste’reactions， resulting in
movement of DNA segments to new sites［Rice and Baker，2001］． It thus leads to
mutations and rearrangements and possibly accelerates biological diversification and
consequently evolution［Aziz et al．，2010］by horizontal gene transfbr［David et al．，
2003］．Therefbre， it is speculated that the hypothetical protein Za10＿0951 is a
transposase that implicates to the cell survival）ility at CHT．
As described in Chapter 2，51the㎜otolerance genes were discovered in E． coll
and classified into 8 groups（A−H）［Murata et al．，2011吻アθ∬］． Interestingly， of tho se 6
groups matched with groups of the㎜otolerant genes in Z脚oわノ1∫5． There is no gene
classified into group A of energy metabolism in Z〃20ゐ∫1’5 genome． This may be due to
the di燈rent of the medi㎜used， LB飴r E． ooll㎝d YPD飴r Z刑o励3． The la廿er
medium contains suf丘cient glucose． Additionaly， genes belong to anti−oxidative stress
response genes飴und in Z吻oわ∫1∫5 which absent in E． co1∫the㎜otolerant gene
categories［Murata et a1．，2011吻γθ∬］．
Interestingly， there is overlapping of the㎜otolerant genes with the genes鉛r
other stresses（Table 3．2）such as the osmotic stress． Overlapping between the genes fbr
the㎜otolerance and genes fbr oxidative stress was鉛md in E． co1∫， but the mechanism
is not adequately understood［G㎜asekera et al．，2008］． Findings in this s加dy would be
usefUl鉛r generating microbes fbr high temperature角㎜entation which expected to the
improvement of production of ethanol or other usefUl materials．
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CHAPTER 4
Physiologic劉l impormnce of cytochrome 6 peroxidase in ethanologenic
thermotolemnt z〃20ゐ’Z菰∫
Abstract
Z脚わ∫1∫3ZmCytC as a peroxidase bearing three heme o−binding motif奮was
investigated with∠Z1η（γC constructed． The mutant exhibited filamentous shapes and
reduction in growth under a shaking condition at a high temperature compared to the
parental strain and became hypersensitive to exogenous H202． Under the same
condition， the mutation caused increased expression of genes fbr three other antioxidant
enzymes． Peroxidase activity， which was detected in membrane丘actions with
ubiquinol−1 as a substrate but not with reduced horse heart cytochrome o， was almost
abolished in∠Zη2のノ’C． Peroxidase activity was also detected with NADH as a substrate，
which was significantly inhibited by antimycin A． NADH oxidase activity of∠」Zη2（γC
was fbund to be about 80％of that of the parental strain． The results suggest the
involvement of ZmCytC in the aerobic respiratory chain via the cytochromeゐcl
complex in addition to the previously proposed direct interaction with ubiquinol and its
contribution to protection against oxidative stress．
4．11ntroduction
Z御oわ∫1／5，aGram−negative and facultative anaerobe in alpha−proteobacteria， is
㎞own to be an ef臼cient ethanol producer［Swings and De Ley，1977；Bara枕i Imd
Buりlock，1986；Thanonkeo，2005］． The organism utilizes the Entner−Doudoroff pathway
［Sprenger，1996］and possesses a relatively simple respiratory chain， consisting of a
type−II NADH dehydrogenase， ubiquinone andろ4−type ubiquinol oxidase［Sootsuwan
et al．，2007］． The activity of NADH dehydrogenase is relatively strong and thus the
respiratory activity may compete fbr NAD（P）H with the ethanol production reaction
under aerobic conditions［Kalnenieks et a1．，2006；Sootsuwan et al．，2007］， and the
respirato塀cons㎜ption of NAD（P）H may limit the reduction of acetaldehyde to
ethanol， pres㎜ably causing acc㎜ulation of acetaldehyde to inhibit cell gro舳
［Viikari and Gisler，1986；Kalnenieks et al．，2008］．
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Since the ethano1飴㎜entation process is exothe㎜ic［Uden，1981；Ghose，
2004］，ethanologenic microorganisms seem to be exposed to heat stress in addition to
other stresses including ethanol［Attfield，1997］and to gxidative stress by endogenous
reactive oxygen species（RO S）including hydrogen peroxide（H202）［Auesukaree et al．，
2009】，which訂e acc㎜ulated more as tempera加re increases［Noor et al．，2009］． H202
causes damage to membrane lipids， cellular proteins and DNA molecules［Condon，
1987；Zagorski，2009］， and such damage influences cell morphology and cell survival in
an additive or synergic fashion［Hartke et al．，1998；Trusca et al．，1998］． Therefbre，
detoxification mechanisms of oxidative stress may be important not only fbr survival of
these organisms but also fbr ethanol production． Several genes responsive to oxidative
stress in Z吻o∂’1∫3 are present in its genome：Z加のノ’C fbr cytochrome o peroxidase，
ZMO1573 fbr iron−dependent peroxidase， Z加cα∫fbr catalase， Z加504 fbr superoxide
dismutase and Z加ψρC fbr alkyl hydroperoxide reductase［Seo et al．，2005］．
Cytochrome o peroxidase（CCP）， which plays an important role in protecting
organisms ffom damage caused by H202， is distributed widely in eukaryotes and
prok田yotes． The structural and fUnctional mderstanding of cytochrome c peroxidase
has’been greatly advanced after the first purification f士om baker，s yeast by Altschul et
al．［1940］． Unlike yeast CCP possessing a singleゐ一type heme as a co−factor， bacterial
CCPs（bCCPs）contain o−type heme． Bacterial genome sequencing， however， has
revealed that bCCP is present in many but not all Gram−negative bacteria and appears to
be absent in Gram−positive bacteria or Archaea． This might be related to the lack of a
periplasmic compartment in these organisms［Atack and Kelly，2007］． Conventional
bCCPs are periplasmic enzymes that contain two covalently bound c−type hemes， thus
called di−heme CCP［FUI6p et aL，1995；Shimizu et al．，2004；Dias et al．，2004；Echalier
et al．，2006］． One of the two hemes fUnctions as the peroxidation site where H202 bihds．
The second heme is the site accepting electrons丘om a physiological donor such as
cytochrome c551 in P5ε短伽oηα30θ70g加050［Hsiao et al．，2007］and cytochrome c2 in
．R乃040ゐααθγ3助αθγo’｛艶8［Abresch et al．，2008］． Interestingly， there are bCCPs with
three heme−binding motifS in E． co1∫and related endobacteria such as G1〃ooηoろαc’θ7
0茂吻η5，Bααθro14θ5加g∫113 and Z励∫113． They bear an N−te㎜inal extension with a
heme c−binding motif（CXXCH）and a putative methionine ligand［Atack and Kelly，
2007］．The tri−heme CCP in オo枷o〃硯γcθ5 αc珈o脚oε’θ刑co加’orη3 has been
demonstrated to be a quinol peroxidase based on evidence that it utilizes reduced
ubiquinone−1 as an electron donor fbr the reduction of H202 to water［Yamada et al．，
2007；Takashima et a1．，2010］．
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Z〃20わ∫1／3cytochrome c peroxidase（ZmCytC）shares homology with tri−heme
CCPs and di−heme CCPs［Atack and Kelly，2007］． Since Z zηoゐ∫1’5 possesses theゐcl
complex and cytochrome o552 without cytochrome c oxidase， ZmCytC is speculated to
accept an electron（s）ffom theわol complex via cytochrome c552（ZMOO961）to convert
hydrogen peroxide to water in the periplasm［Sootsuwan et al．，2007］． However， no
direct．evidence fbr this speculated electron transfbr process has been provided．
In this study， we constructed a㎞ockout mutant of Z加（ッ’C to elucidate its
physiological fUnction． Effbcts of mutation on growth and morphology and on the
expression of genes fbr other antioxidant enzymes were examined． Biochemical
analysis was also per鉛㎜ed to dete㎜ine whether ZmCytC catalyzes peroxidase
reaction with reduced ubiquino1−10r NADH as an electron donor in membrane
丘actions． The NADH−dependent peroxidase activity was inhibited by the addition of
antimycin A． On the basis of these results， we discuss the physiological flmction of
ZmCytC and its possible electron donor in Z〃20ゐ∫1∫5．
4．2Materials and methods
4．2．1Materials
Restriction enzymes and T4 DNA ligase were purchased丘om Takara Shuzo
（Kyoto， Japan）and New England Biolabs． A DNA sequencing Kit（ABI PRISM⑭
Te㎜inator v 3．1 Cycle sequencing Kit）was obtained丘om Applied Biosystem Japan．
Oligonucleotide primers（Table 4．1）were synthesized by Proligo Japan K．K．（Tokyo，
Jap㎝）． Other chemicals were all of㎝al外ica1匹ade㎝d obtained丘om co㎜ercial
sources．
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Table 4．1 Primers used in this study
望 ’
P血ners
Seqμen◎e（5暫to 3）
cytC−5
CtCtgaaagCCagaaCC
cytC−up−kan−3
CagCtCCagCGtaCaCagaggttgttgCCCCata
cytC−down−kan−5
aaggaggatattCatatgCtatCgCggCCataatC
cytC−3
tCgCCatCaaaagCaCg
Kan・c｝忙C−5
tatg999CaaCaaCCtCtgtgtaggCtggagCtg
Kan−cytC−3
gattatggCCgCgatagCatatgaatatC（えCCtt
cytC・5Rr
CttCtgtgatCgttgCC
cytC・3Rr
aaaggtCtatggCggtC
sod−5RT
CCCagaCatCgagCgtC
sod・3RT
taCCCgCCCtgCCCttt
cat−5RT
gCCCaaCCCtCCt㏄aa
cat−3RT
atgCCCtgttgtgaCag
ahpc−5RT
gCaag99CaaaggtCat
ahpc−3RT
gttCagCag99tgtatC
丘on−5RT
CCgaaCCCCtgCCCCaa
廿on−3RT
tCttCaCCatCCCgαC
TAIL−840up l
CCaagaCagaaagCaCatCC
TAIL−840up2
CgCtgtCatatCgCCat
、 ’マ 、気㌧
4．2。2Bacterial strains， culture conditions and membrane preparation
Bacterial strains and plasmids used in this study are sho㎜in Table 4．2 Z
刑oわ∫1∫3TISTR 548 and its mutant，∠Z駕の7∫C， were grown in YPD medium consisting of
O．3％yeast extract，0．5％peptone and 3％glucose［Michel et al．，1985］at 30°C or
37°Cunder a shaking condition at 2001pm or㎜der a static condition［Taherzadeh et
al．，1997］． Cells cultured fbr g h and 24 h（corresponding to optical densities of about
O．8㎝d2．5 at 550㎜㎜der the shaking condition飢1d about 15 and 3．O at 550㎜
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under the static condition， respectively）were used fbr some experiments as those in
exponential and stationary phases， respectively． To prepare membrane f士actions， cells
were harvested and suspended in 10mM potassium phosphate buffbr（pH 7．0）and then
passed twice through a French press at 16，000 psi， fbllowed by centrifUgation at 86，000
gfbr 90 min［Sootsuwan et al．，2007］． The pellet was then homogenized with the same
buffbr and used as a membrane ffaction．
Table 4．2 Bacterial strains and plasmids used in this study
Stf血Qr plas卿d
Gehotype
Re免等n◎e ビ 、ミ
r 文
ヂ ヘニ E．oo1∫DH5α
F一 Ψ80∠11αoZ∠M15、」（1αoZZ4一醐g戸）
・馬 ぎ；¶
Novagen
び169卸朗1θ醐1加御7（rk’， mk＋）
p乃α45z卯E44λ’功’lgy〃196彫ゐ41
Z〃20ゐ∫1∫εTISTR 548
Wild type
TISTR
Z吻oゐ’1∫3T548』cytC
Z．脚oわ∫1∫5TISTR 548△Z加￠y‘C
Present work
Amp「
Promega
Plasmid
pGEM−T Easy
pDK4
pGEMZMCYTC：：Km
Km「
K．A． Datsenko
pGEM−T Easy carryhlg a廿agment
Present work
of3．4−kb；sequence upstream and
downstream kb ofZ功｛卯C with
insertion ofl5−kb ofkanamychl
reSIStanCe gene
pZA22
tet「 ， cml「
H．Yεmase
pZAZMCYTC
pZA22 carrying a 3．5−kb食agment of
Present work
the fUll Iength ofZ加のぜC gene
between theβα吻HI and 5α110f
pZA22
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4．2．3DNA m紐nipul紐tion
Conventional recombinant DNA techniques were applied［Sambrook and
Russell，2001］． To construct an expression plasmid of Zンηのノ’C， we designed primers
（Table 4．1）specific to the region about 840 bp upstream丘om the ir直tiation codon of
Z加（ζソ’Cand the region 1，100 bp downstream丘om its termination codon， on the basis of
the genome sequence of Z溺o∂∫1∫3 strain ZM4 strain［Seo et al．，2005］． Using a primer
set of cytC−5 and cytC−3， a 3，462−bp DNA ffagment was amplified by PCR with
PrimeSTAR（TAKARA BIO INC， Shiga， Japan）and Z吻oゐ∫1∫5 TISTR 548 genomic
DNA as a template． The amplified DNA ffagment was cloned into pGEM−T Easy． The
construct was血曲er con且㎜ed by nucleotide sequencing［Sanger et al．，1977］． The
plasmid DNA was digested with jBαηzHI and 8α11， an4 DNA丘agment containing
Z加（γCwas ligated into the、Bα吻HI−8α11 site of pZA22， generating pZAZMCYTC． A
Z加のκ一disrupted mutant strain was constructed by homologous recombination． The
recombinant DNA ffagment fbr the homologous recombination was prepared by fUsion
PCR［Kuwayama et al．，2002］． The DNA f士agments of upstre㎜and downstream
sequences of Z〃2（γC were separately amplified by PCR using Zη20ゐ’1∫5 TISTR 548
genomic DNA as a template and a primer set of cytC−5 and cytC−up−kan−3 and a primer
set of c》柁C−do㎜一kan−5 and c外C−3， respectively． PCR reaction was perfb㎜ed with as
fbllows：heating to 98°C fbr l min and 30 cycles of 98°C fbr lO s，52°C fbr 10 s and
72°Cfbr 2 min， fbllowed by a final extension at 72°C fbr 5 min． The kan㎜ycin
resistance cassette（肋η）was amplified by PCR using pKD4 plasmid DNA carrying the
肋ηgene as a template with a primer set of kan−cytC−5 and kan−cytC−3． PCR products
were separated on O．9％agarose gel． The corresponding DNA bands were excised and
purified with Easy trap（TAKARA BIO）． The Fusion PCR with the three purified DNA
丘agments was perfb㎜ed using a PuReTaqTM Ready−To−GoTM PCR beads kit（GE
Healthcare， Buckinghamshire， UK）as fbllows：94°C fbr l min and 10 cycles of 94°C
fbr 10 s，40°C fbr 10 s and 72°C fbr 4 min， fbllowed by an extension at 72°C fbr 5
min． After the process，10 pmol primers of cytC−5 and cytC−3 were added to the
reaction tube and the fUsion was restarted as fbllows：94°C fbr l min and 30 cycles of
94°Cfbr 10 s，52°C fbr 10 s and 72°C fbr 4 min， fbllowed by an extension at 72°C
fbr 5 min． The血sion PCR product was then purified and ligated into pGEM−T Easy．
The resultant clone was verified by nucleotide sequencing． The deletion in the Zンηの1∫C
ORF of the clone was 1，355 bp encompassing丘om the 23「d codon to the 452nd codon．
The plasmid DNA was isolated and introduced into Z．1ηoゐノ1∫5 TISTR 548． The
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disrupted mutants were screened on YPD agar plates containing kanamycin（50μg／ml）．
The Z加げC disruption was confirmed by using the genomic DNA ffom the mutant as a
template and fUrther proved by digestion with P3∫1，0f which a recognition site is present
in theんαηgene． F賦he㎜ore， the speci且c inse丘ion of the recombin｛mt仕agment
including theんαηcassette into the genome was examined by using TAIL−PCR［Lui et
al．，1995］fbllowed by the nucleotide sequencing， which dete㎜ined sequences outward
丘om the 840−bp upstream position丘om the initiation codon of Z瓶γC and sequences
outward ffom the 1，100−bp do㎜stream position丘om the stop codon． Briefly， the
mutant genomic DNA used as a template fbr l st TAIL−PCR with a specific primer of the
upstre㎜TAIL−840up l or the do㎜stream TAIL−1100do㎜10f Z吻（ッ’C and arbitr町
primer（AD20r AD3）． The lst PCR products were used as the template fbr the 2nd
TAIL−PCR with a specific primer of the upstream TAIL−840up20r the do㎜stream
TAIL−1100down20f Z加の1’C and arbitrary primer（AD20r AD3）． The 2nd PCR
products were used as the template fbr the 3「d TAIL−PCR with a specific primer of the
up stre㎜TAIL−840up30r the downstre㎜TAIL−1100down30f Z加のノ’C and arbitrary
primer（AD20r AD3）， all of the TAIL−PCR ampli且cation was perfb㎜ed as described
previously［Lui et al．，1995］． The 2nd PCR product or 3「d PCR product was su切ected to
nucleotide sequencing on an ABI PRISM310（PE Biosystems）with a primer of
TAIL−840up 30r TAIL−1100down3
4．2．4H劉lo ass紐y
Halo assays fbr sensitivity to H2020f the∠Z吻（γC and its parental strains and
their transfb㎜ant with pZAZMCYTC or｛m empty vector were perfb㎜ed as described
previously［Lertwattanasakul et al．，2009］． Briefly，1ml of culture at mid−exponential
phase（density to OD550＝・1）was mixed with 2 ml of soft YPD agar（0．8％agar）and
spread on YPD ag訂plates． V肛ious vol㎜es of 10％（v／v）H202 were placed on the
center of the sterilized discs． This paper disc assay was carried out in triplicate cultures
fbr each strain and the data were treated statistically．
4．2．51ntracenular ROS leve1
An oxidative−sensitive probe， dichlodihydrofluorescein H2DCFDA， was used
to measure the intracellular ROS leve1［Lertwattanasakul et a1．，2009］． Briefly， cells
were grown in YPD medium with and without 10μM H2DCFDA at 30°C or 37°C
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under a shaking condition at 200 rpm fbr 24 h． Cells were then harvested and washed 3
times with 10 mM potassium phosphate buffbr（pH 6．8）． They were then su切ected to
either observation of cell morphology or measurement of fluorescent intensity after
disruption by so㎡cation and removal of cell debris． The fluorescent intensity was
meas肛ed using a Hitac㎞650−10S Fluorescence spectrophotometer（excitation，504㎜；
emission，524㎜）． Emission values were no㎜alized by protein concentration．
4．2．6RT−PCR analysis
Cells were gro㎜in YPD medi㎜鉛r 24 h， and total剛A was i㎜ediately
extracted using the hot phenol method［Aiba et al．，1981］． To examine the expression of
Z加（γC（ZMO 1136）， Z加傭（ZMOO918）， Z加504（ZMO 1060）， Z脚勿C（ZMO 1732）
and ZMO1573， RT−PCR was perfb㎜ed using an mRNA−selective RT−PCR kit
（TAKARA BIO）， two specific primers fbr each gene and O．1μg of total RNA as
described previously［Tsunedomi et al．，2003］． The primer set used fbr each gene is
shown in Table 4．1． After RT reaction had been perfb㎜ed at 40°C fbr 15 min， PCR
consisting of denaturing at 85°C fbr l min，45°C fbr l min and extension at 72°C fbr
2min was carried out． The PCR products after 20，25，30 and 35 cycles fbr each gene
were taken and analyzed by O．9％agarose gel electrophoresis， fbllowed by staining with
ethidium bromide． Intensity of bands of RT−PCR products was quantitatively
dete㎜ined using the UN−SCAN−IT gelTM auto甑ed digitizing system（Silk Scienti且c）．
Linearity of the amplification was observed up to the 25th or 35血cycle． Under our
conditions， the RNA−seledtive RT−PCR was able to specifically detect mRNA because
no band was observed when reverse transcriptase was omitted．
4．2．7Analytical procedures
Ethanol and glucose concgntrations in the medi㎜were measured by high
per鉛㎜ance liquid chromatography（HPLC）（Hitachi Chemical， Japan）using Gelpack
GL−C610−S（7．8 x 300㎜）． The medi㎜丘action was prep飢ed as a supema伽t a丘er a
low−speed centrifUgation of Zη20ゐ∫1∫5 cultures at diffbrent cultivation time points and
diluted 10 times befbre applying to HPLC． Using HPLC with a reflective index
detector， chromatographシwas per飴㎜ed at 60°C and degassed water was used as a
mobile phase at a flow rate of O．3 ml／min． The concentration was then compared with
the standard of㎞o㎜concentrations of ethanol or glucose．
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Ubiquinol and NADH oxidase activities were measured spectrophotometrically
at 25°C by鉤llowing the decrease in absorbance at 275㎜and 340㎜， respectively
［Eliasθ∫α1．，2001］． One㎜it ofubiquinol−1 and NADH oxidase activities is de且ned as 1
μmol of ubiquinol−l or NADH oxidized／min， which was calculated with a millimolar
extinction coef丘cient of 12．25 fbr ubiquinol and 6．25 fbr NADH． Peroxidase assay was
carried out as described previous［Yamada et al．，2007］． To measure ubiquinol or
NADH peroxidase activi取， the cons㎜ption of uわiquinol or NADH was dete㎜ined in
the assay mixture as fbr the ubiquinol or NADH oxidase expect fbr the addition of l
mM glucose and 50㎜it glucose oxidase． The reaction was initiated by the addition of
10mM H202 to the final concentration of O．01 mM． The cytochrome o peroxidase assay
was perfb㎜ed at 25°C in 100 mM Tris−HC1（pH 7．5）and 30μM horse hea式
cytochrome o［Yamada et al．，2007］． The reaction was ir直tiated by the addition of 10
mM H202． Oxidation of c算oc㎞ome c was measured翫550㎜．
4．2．8Database search and computemm藍ysis
Database searching was perfb㎜ed with DDBJ， NCBI， Swiss−Prot and TrEMBL
databases． Comparison of primary sequences， hydropathy plot analysis and multiple
alig㎜ents were conducted by using GENETYX（So負w肛e Development， Tokyo，
Japan）．
4．2．9Statistics
Unless otherwise indicated， mean and SD calculated丘om data of at least three
independ弔nt experiments are・presented． The variations between the experiments were
estimated by SD， and the statistical significance of changes was estimated by
two−sample’−test assuming equal variances．＊＊＊，＊＊and＊mean p＜0。001，＜0．01 and
＜0．05，respectively．
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4．3Resuhs
4．3．1Strmctural characteristics of ZmCytC
Comparison of the primary structure of ZmCytC， which is deduced丘om its
nucleotide sequence， with prim町structures of other bCCPs was perfb㎜ed（Figure
4．1）．The length of the amino acid sequence of ZmCytC corresponds to those of
tri−heme c peroxidases and about 100 amino acid residues including one heme o−binding
motif extend at its N−te㎜inus compared to sequences of di−heme CCPs． Hydropathy
analysis revealed that there is one possible membrane−spanr血g segment at the
N−te㎜inal（data not shown）， which is responsible fbr associati6n with the inner
membrane． Although the N−te㎜inal about 20一㎜ino acid sequence has a character of
basic amino acids fbllowed by a hydrophobic sequence， similar to that of a signal
sequence， it may be not removed because a similar sequence is present in purified
tri−heme CCP inオ．αc加o〃2ッcθ’θ吻co砺如η3［Yamada et al．，2007；Takashima et al．，
2010］．This is consistent with the finding that most peroxidase activity in Z脚oゐ∫1’5 was
recovered in membrane丘actions（see below）． ZmCytC shares 39−43％sequence
identity with di−heme CCPs and 44−57％with tri−heme CCPs． Yamada et al．［2007］
reported that the tri−heme CCP inオ．αc珈oη宅ycε∫θ〃2co〃2∫’oη3 is ubiquinol peroxidase，
which is very similar in primary sequence to ZmCytC（47％identity）． These findings
strongly suggest that ZmCytC is a member of a tri−heme CCP￡㎜ily． However， since it
o
has been annotated as a cytochrome c peroxidase in a database， it is also important to
clarifンthe electron donor fbr ZmCytC．
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ZrnCytc
GdiCCP
EGOCCP
AaGCCP
RCPCCP
RSPCCP
PaeCCP
ZrnCytc
GdiCCP
EcoCCP
AacCCP
RCPCCP
RSPCCP
PaeCCP
1
1
1
1
1
1
55 叩Ω￥9耳TPN互E亀P−FXA興LvGs肌QNplKE6NRvFLLNELエNGLKDPsKLsEvD弄AKL
117
117
116
113
1
1
1
1
1
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59 RqDぎCHTKDA既PF−￥ASLPVAKQLMQ吻・KRgLQH穿Q・Ω肌N袖EKG巳AVDEES恥脳
59 RCDYρ甑朗T肌PF−YFH甲ANQLMQ即VDQgLR町RエEPVL簡FQSGAVPSEεQμRI
58
GCDμTPSA肌PAYY−YI戸GAKQLMDYDエKLGYKSドNLEAV㎜LADKPVSQSDゐNKI
116 Q即EHD剛PP囮YLT騨”二GEKGK磁E野工KES琴IKNYD皿GLVAPQドΩS甲喫
ZmCytc
GdiCCP
177
176
175
171
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PVPET−LPVDA㎜工GQ一
KQ
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41
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翼、是。：
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ZmCytc
GdiCCP
EcoCCP
AacC（：P
RcpCCP
RspCCP
PaeCCP
ユ18
ユ17
1
18
351
351
349
343
219
214
195
ZmCytc
GdiCCP
EcoCCP
410
411
406
403
276
271
252
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GdiCCP
EcoCCP
AacCCP
RCPCCP
RSPCCP
PaeCCP
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．HD解GVADKLKQDPT誕LT
じ．1（S興DEエエAKLEKDPΩ工KT
G§HS胆BエVARFEΩDEDFKQ
凶国TP巳N工VATVQSMPG一ヨVE
TPEΩVERTLRSMPΩ一押E
細PQLVEΩTLGSエPE噂VD
96
76
ZmCytc
GdiCCP
EcoCCP
AacCCP
RcPCCP
RspCCP
PaeCCP
RcpCCP
RSPCCP
PaeCCP
174
170
DA工HDΩASAL一写KPIPEΩ
一一一一一一一一一一一一一一一一一一一一一一一一一一一一一一一一一一一一一一一一一一
234
233
232
228
100
294
293
292
288
159
155
135
A己cCCP
ユ76
ユ75
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E㎜ΩyET贈PTRYT肌興6K−VSDEE麗1工螺碕KQ興EYYASNDTAPEH剛EPVQ『
114 ERVIENDE腱工AKF］」HI剛GSR−PDEDEKTA工LN吼REQRKAFLPVNTEGTDNHRLV−Q−
一一ニー一一一一：：…−MK−一長TgエSLエAVAT上L訟P塩ED五一一A−L肥BAKGL乖V工麺
1
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GdiCCP
ECOCCP
AacCCP
RcpCCP
RSPCCP
PaeCCP
Z皿Cytc
57
54
lMKKFALKTAWAI−−VG−−Y工GWG㎜QYDKGQMEKLLAE6SYSAEQQ−KIA即FFNN−
ZmCytc
GdiCCP
EcoCCP
AacCCP
RcpCCP
RspCCP
PaeCCP
AacCCP
RCPCCP
RspCCP
PaeCCP
58
58
1 貿NIKA工FGSAV▽LA▽▽A−−YGATTSYLVHYDHETAPKLSS工SPTNNNPKAKAAFDTIAEA
l MSVRKLVlSVAALGCVA−−1「GGTVGYLTHFDHDTAPTLGTNSPT五ADPVASAAF瓦AIRES
l b5KMVSR工TA工G−LAGVA工C￥LGISG㎜一YHDNKRSKΩADVQASA▽SENNKVLGF−LREK
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232
231
227
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95
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293
292
291
287
158
154
134
350
350
348
342
218
213
194
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410
405
402
275
270
251
464
465
459
456
334
329
310
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473
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330 ］」PPETASTPRPTAEVKVD
311 ］」PASTETTPRPAE−一一一一
弓65
460
349
347
323
Figure 4．l Alignment ofdeduced amino acid sequences ofZmCytC and other bCCPs．
Deduced amino acid sequences of ZmC）紘C丘om Zη20ろ∫1∫3（NCBI：ZMOll36），
GdiCCP ffom G1μcoηασαoわααεr読αzo’rqρ毎cμ5 PAl5（NCBI：Gdia O285）， EcoCCP
丘om E oo1’（NCBI：AAC76543．1）， AacCCP丘om．4．αc∫oηo燗ッcθ∫θ配oo纏’αη5（DDBJ：
AB269691）， RcpCCP丘om R乃．σ￠ρ∫㍑1α砲3（DDBJ：RCAP rccO l 723）， RspCCP丘om
R海5ρ加εアo’（左3（DDBJ：Rsphl7025＿1377）and PaeCCP fヒom Pαεrμg加05αbCCP
（PDB：lEB7）were aligned． The parameters were as fbllows：gap open＝10， gap
extension＝0．05， and gap distance＝8． Conserved residues are shadowed． Numbers to
the right denote residue positions． Three heme o−binding motif9（CXXCH）are indicated
by thick upper lines． ZmCytC， GdiCCP， EcoCCP and AacCCP have 3 heme o−bindhlg
motif忌and other three CCPs have 2 heme c−binding motifbs．
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4．3．2Effbct50f disruption or increased expression of Z配の1’C on morphology，
growth紐nd ethanol production
In order to clarifンthe physiological fUnction of ZmCytC， we constructed a
disrupted mutant of Z加（γC by homologous recombination with a肋ηcassette． The
speci且c disruption of the gene was con且㎜ed by TAIL−PCR fbllowed by nucleotide
sequencing， which dete㎜ined the outward sequences both就upstream and do㎜stream
junctions between the genome and the DNA丘agment inserted fbr the ZmCytC
disruption． Only a single sequence was fb㎜d at each j㎜ction， suggesting that the
insertion of the DNA丘agment including the肋ηcassette was only one place in its
chromosome． Z脚ろ11∫∫TISTR 548 used in this study ls the㎜otolerant and it can grow
Imd produce ethanol even at a relatively high temperature㎜der a shaking condition
［Sootsuwan et al．，2007］and㎜der a static condition（mpublished）． We thus examined
growth and molphology of∠Z雁ッ’C at 30°C and 37°C because it was expected that
cells would be exposed to more oxidative stress as temperature increased［Noor et al．，
2009］．When ROS in TISTR 548 was observed by a specific fluorescent probe，
dichlodihydrofluorescein H2DCFDA， its level at 37°C was about 1．7−fbld higher than
that at 30°C under shaking conditions， but hardly changed under static conditions（data
not shown）．
No significant diffbrence in colony morphology between∠Z吻のノ’C and its
p飢ent was observed on YPD plates or in liquid medi㎜under static conditions． We
thus examined the effbcts of the mutation on growth and morphology in liquid medium
under a shaking condition． The growth curves（Figure 4．2A）revealed that the Z初の4C
mutation caused negative effbcts on growth at 37°C and 39°C， but not at 30°C．
Morphological observation showed that both Z加（ッ’C mutation and parental cells
bec㎜e eIongated at 37°C and 39°C（Figure 4．2B and data not sho㎜）and the mutant
cells were a little longer than the parental cells．4’−6−Di㎜idino−2−phenylindole（DAPI）
staining exhibited a line of nucleoides along with elongated cells at high temperatures
（data not shown）， suggesting that the elongation is due to the inhibition of cell division．
Even at 30°C， the average cell length of mutant cells was 1．3±0．2−fbld longer than that
ofthe parental cells．
We fU曲er attempted complementation experiments with a plasmid clone of
Z加（γC（Figure 4．2C and D）． However， introduction of an empty vector， pZA22， was
fbund to cause a large reduction of growth at 37°C though no such effbct was fbund at
30°C．We have no clear reason fbr this negative ef飴ct of the plasmid． So， due to the
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ef〔bct， no complementation by pZAZMCYTC was observed in growth at 37°C（Figure
4．2C）． Cells may require more energy to maintain plasmid molecules at a high
temperature． With regard to cell morphology， the introduction of pZA22 caused
elongation at 30°C（Figure 4．2D−b and F−b）． Both∠Z泌（γC bearing pZAZMCYTC and
the parental strain bearing pZAZMCYTC were fbund to be shorter in cell size than
those bearing an empty vector， pZA22 both at 30°C and 37°C， indicating that cell
elongation was suppressed by the addition of pZAZMCYTC．
We㎞her ex㎜ined the e館ct of the Z卿C mutation on glucose cons㎜ption
and ethanol production at 30°C and 39°C㎜der static and shaking conditions（Figure
4．3）．Under static conditions， glucose consumption and ethanol production were faster
at 39°C than at 30°C． No significant effbct of the mutation， however， was observed．
Under the shaking condition， glucose consumption was much less at 39°C than at
30°C，probably due to repressed growth at a higher temperature（Figure 4．2A）． At
30°C，the∠Zη2（γC showed 23％and 40％increases in ethanol production at 24 h and
48h， respectively， compared to those of its parent（Figure 4．3C）． These increases may
be due to the increase in amount of NADH available fbr ethanol synthesis since NADH
oxidase activity was reduced in the mutant（see below）． Taken together， the results
suggest that Z加のノ’C is crucial fbr growth at a high temperature．
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15
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o5
0．o
12
T㎞e（h）
z5
2，0
L5
1．o
0．5
臥o
豊2
Time（h）
25
zo
1．0
胚
12
Time（h）
Figure 4．2 Growth curves and morphology ofZ加の∼’C mutant and parental strains under
ashak㎞g condition and effbcts ofZ功（ツ’C clone．
Cells were grown in YPD medium under a shaking condition（200 rpm）at difibrent
temperatures and cell morphology at 24 h was observed． A Growth curves of Z功のノ∫C
mutant（opened symbols）and parenta1（closed symbols）strahls at 30°C（ch℃les），37°C
（triangles）and 39°C（squares）． B Cells morphology of Z加（ッ’C mutant（B−a）and
parental strain（B−c）at 30°C and ofZ功（ッ’C mutant（B−b）and parental strain（F−d）at
37°Cin figure A． C Growth curves of Z次ッ∫C mutant／pZAZMCYTC（circles）and
Z加（γCmutant／empty vector， pZA22（triangles）at 30°C（closed sylnboIs）and 37°C
（open symbols）． D Cells morpho logy ofZη3（γC mutant／pZAZMCYTC at 30°C（D−a）
andき7°C（D−c）and Z1ηの2∫C mutant／pZA22 at 30°C（1）−b）and 37°C（1）−d）in figure C
EGrowth curves of the parental strain／pZAZMCYTC（diamonds）and the parentaI
stra血〆pZA22（squares）at 30°C（closed symbols）and 37°C（open symbols）． F Cells
morphology of the parental strain／pZAZMCYTC at 30°C（F−3）and 37°C（F−c）and
the parental strain／pZA22 at 30°C（F−b）and 37°C（F−d）血figure E． Pllotos shown in
B，Dand F are also representatives． Scale ba1，10μm．
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Figure 4．3 Ef〕驚ct ofdisruption ofZ加のノ∫C on glucose utilization and ethanolproduction．
Cells were grown in 30 ml of YPD medium at 30°C（A）or 39°C（B）under static
conditions and 30°C』（C）or 39°C（D）under a shaking condition（200 rpm）． Gluco se
and ethanol concentrations were dete㎜㎞ed at the time㎞dicated． Open and closed
circles represent glucose concentration in the medium of the Z加の2∫C mutant and
p肛ental stra加s， respectively． Open㎝d closed colu㎜s represent ethanol concentr翫ion
on the Z加（卯C mutant and parental strahls， respectively．
4．3．3Sensitivity to H2020f a disr叩ted mutant ofZ肱ッκ
Since Z襯卿C was expected to encode cytochrome c peroxidase， the sensitivity
to H2020f△Z濯のノ’C was examined on plates containing filter discs that absorbed
diffbrent volumes of 10％H202（Figure 4．4A and B）．∠1Z配のノ’C showed halos larger than
those of the parental strain， suggesting that the mutation caused cells to be
hypersensitive to H202． The effbct of Z加のノ’C on sensitivity to H202 was fUrther
examined by introduction of pZAZMCYTC into∠Z珊ゆC（Figure 4．4C）and its parent
（Figure 4．4D）． In the case of∠Z雁γC， the introduction of the plasmid clone greatly
reduced the halo size compared to the control strain harboring pZA22． Similar but
slightly smaller effbct was observed in experiments with the parental strain． These
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results suggest that ZmCytC contributes to the protection against oxidative stress．
Notably；comparison of hal6 sizes at the same concentration of H202 in Figure 4．4B， C
and D revealed that introduction of even an empty vector caused hypersensitivity to
H202． Maintenance ofthe plasmid might be a burden to cells．
30
曾
δ20
75騨110μ125蝉
H幽（1096v幼
ε・・
ε
820
75解1 10四虚】L25圏畷 15四
75聾1mμ112．51直15μ蓋
Hi防（10％卿）
Hρ2（10％▼《り
Figure 4．4 Effbct of disruption or㎞creased expression of Z1ηのノ∫C on sensitivity to
exogenous H202．
Disc diflUsion assay was perR）rmed on YPD plates at 30°C as described in
Exper㎞ental Procedures． Typical examples are shown㎞A． Halo diameters detemined
were shown in B fbr the Z加（ッ∫C mutant（open columns）and parental（closed columns）
strains， in C fbr Z雁γC mutant／pZAZMCYTC（open columns）and Z溺（ッ’C
mutant／pZA22（clo sed colu㎜s）and血D偽r the parental strai㎡pZAZMCYTC（open
columns）and the parental stra㎡pZA22（closed columns）． Reported values are the
mean（±SD）ofthree hldependent experiments． p values were calculated by two−sample
∫−teSt aSSUming eqUal VarianCeS．
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4．3．4Expression of genes presumably related to oxid3tive stress response and
ef6ect of a disrupted mutation ofZ肱ッ’C on their expression
On the basis of the fUnction of ZmCytC in protection of oxidative stress as
described above， it was ass㎜ed that the Z加g∫C dismption a館cted the expression of
genes related to oxidative stress response， Z〃2cα∫， Z加504， Z駕α勿C and ZMO1573． The
ass㎜ption was ex㎜ined by RT−PCR with total RNA prep肛ed丘om cells that were
gro㎜in YPD at 30°C and 37°C㎜der static or shaking conditions（Figure 4．5）． The
band intensity in each PCR cycle corresponded to the level of mRNA examined． The
intensity of RT−PCR products of Z加（ッ’C in the parental strain was stronger at 37°C
than that of 30°C㎜der both static and shaking conditions and was stronger under the
shaking condition than㎜der the static condition at both temperatures． There was no
band in∠Z刑（ッ∫C， confi㎜ing disruption of the Z吻∫C gene． The intensity of products
of ZMO1573 was also stronger at a higher temperature㎜der the shaking condition．
Therefbre， Z加の4C and ZMIO1573 are thought to be up−regulated at a higher
temperat皿e， a condition in which oxidative stress is more acc㎜ulated． On the other
hand， the results suggest that Z加oo’is do㎜一regulated at a high temperature．
Next， the effbct of the Z脚（γC disruption on expression of Z初504 Z初cα乙
Z加α勿Cand ZMO1573 was examined by comparison of the intensities of RT−PCR
products f士om the mutant and the parent． A significant diffヒrence in the intensity was
observed in Z加吻C and ZMO1573㎜der the shaking condition at 37°C and in Z加504
and Z加α勿C under the static condition at 37°C． These results suggest that Z加504
Z加α勿Cand ZMO1573 are complementarily expressed to the Z加のノ’C mutation．
ZmCytC may thus be crucial fbr the protection of Z．1ηoわ∫1∫5丘om oxidative stress，
especially at a high temperature．
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B
Cycles
Static，30°C
Shaking，30 C
Parent △Zη2のノ祀
Parent △Z〃κツ記
25 30 35 20 25 30 35
20 25 30 35 20 25 30 35
Z加のノ∫C
Z加∫04
Z加oα’
Z加1ψC
ZMO1573
16∫rR八乙4
D
Cycles
Static，37 C
Shaking，37 C
Parent △Z〃2（ツκ⊃
Parent △Z加のノごC
25 30 35 20 35
20 35 20 25 35
Z吻（ly￡
Z〃20α∫
Z加α1ψC
ZMO1573
163r㎜
Figure 45 Expression ofZ加（γC， Zη2∫04 Zη20厩， Z功α勿ワC and ZMO1573 under static
and shaking conditions at 30°C or 37°C．∠1Z溺の2∫C and the parental cells were grown in
YPD medium at 30°C（A）or 37°C（B）under a static and shaking condition（200 rpm）
at 30°C（C）or 37°C（D）R）r 24 h， f｝om which total RNA was prepared． RT−PCR was
then carried out with O，1μg oftotal RNA except that O．001 Fg oftotal RNA was used
fbr 16S rRNA， and PCR products f｝om 20，25，30 and 35 cycles were analyzed by
agarose gel electrophoresis． E Relative band intensity of bands in A−1）was dete㎜ined
as described in Experimental Procedures。
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4．3．5Peroxidase 3ctivity of ZmCytC and its possible electron donor
Peroxidase activities of the」Z〃7（γC and parental strain in membrane ffactions
were compared（Table 4．3）． Ubiquinol−1 peroxidase activity was detected in the parental
cells， but only a trace of amount of ubiquinol−1 peroxidase activity was detected in the
mutant cells． NADH peroxidase activity in the mutant was also significantly lower than
that in the parental cells． These results suggest that ZmCytC fUnctions as a peroxidase in
the inner membrane and receives electrons ffom the respiratory chain．
Zη20ろノ1∫5possesses a simple respiratory chain consisting of dehydrogenase，
ubiquinone， and ubiquinol oxidase［Sootsuwan et aL，2007］． The organism， however，
retains the cytochromeわol complex and possibly its electron acceptor as a cytochrome
c，though there is no c）花ochrome c oxidase in its genome． We thus examined whether
ZmCytC accepts electrons via the cytochromeわol complex， by using antimycin A as an
inhibitor of cytochromeわ01 complex． The addition of antimycin A greatly reduced
NADH peroxidase activity in the parental strain． The remaining activity was neafly the
s㎜eas the level of NADH peroxidase activity in、」Z〃2（ッ’C． On the other hand， the
activity of ubiquinol−1 peroxidase was hardly inhibited by antimycin A， and it is
therefbre likely that the cytochromeわcl complex fUnctions as the electron donor fbr
ZmCytC．
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4．4Discussion
ZmCytC in Z溜oわ’1∫5 has been a㎜otated as c）荏ochrome c peroxidase［Seo et
al．，2005］， though it seems to possess three heme c−binding motif忌similar to ubiquinol
peroxidase in．4．αo伽o〃尾yoθ∫θ加co加1αη3［Yamada et a1．，2007；Takashima et a1．，2010］．
In order to dete㎜ine the physiological㎞ction of ZmCytC and its electron donor，
Z加（ワ1C−disrupted mutant was constructed and examined． Our findings suggest a unique
strategy of the organism to protect cells against H202 in association with respiratory
chain
CCP and ubiquinol peroxidase are thought to contril）ute to protection against
exogenous oxidative stress． The Z加（ッ∫C mutant was sho㎜to exhibite more sensitivity
than the parental strain to exogenous H202（Figure 4．4）and reduced growth at a high
temperature under the shaking condition（Figure 4．2）． Since such a growth defbct was
obvious under，七he shaking condition but not under the static condition， the defbctive
phenotype may be due to the critical level of endogenously generated H202， which was
suggested to be acc㎜ulated more at a high temperature． The肋げC mutant and
parental strains became filamentous under the shaking condition at high temperatures．
This significant morphology may be due to inhibition of cell division as observed when
DNA damage is acc㎜ulated． H2020xidizes solvent−exposed iron， resulting in the
鉛㎜ation of hydroxyl radicals which severely damage DNA molecules．1血such a
situation， cells stop cell dividing and become filamentous［Zagorski，2009］． The Z脚の1’C
mutation caused an increase in the expression of genes fbr antioxidant enzymes， Z廊04，
Z功碗ρCand ZMO1573， especially at a high temperature． Such a response， however， did
not fUlly suppress the Z加のκmutation under the shaking condition at a high
temperature． These finding and the up−regulation of Z吻（γC at a high temperature
suggest that ZmCytC as a H202−degrading enzyme is crucial fbr survival of Z脚ゐ∫1∫5 at
ahigh temperature．
Stmctural comparison with other CCPs including the extensively analyzed
ubiquinol peroxidase ofオ．αc伽o剛cθ∫θ刑coz漉αη5［Y㎜ada et al．，2007；Takashima et
al．，2010］suggests that Z溺o励5 ZmC）控C is a membrane−bomd pero琴idase with three
heme o−binding motif忌． ZmCytC−dependent peroxidase activity was detected in
membrane丘actions when ubiquinol or NADH， but not reduced horse heart cytochrome
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c，was used as an electron donor． SuΦrisingly， NADH peroxidase， but not ubiquinol
peroxidase was inhibited by anヰimycin A， which is㎞o㎜to be an ir雌bitor fbr the
c）沈ochromeろ01 complex（Table 4．3）， suggesting that theわcl complex mediates the
electron transfヒr f士om NADH to ZmCytC． Cytochrome c552 bearing a putative signal
sequence is expected to be an additional mediator［Sootwan et al．，2007］． If this is the
case， electrons f士om the cytochromeろcl complex via cytochrome o552 are fbd by
ZmCytC to H202 in the periplasm Considering the report that purified tri−heme CCP in
．4．oc珈o〃竃γcθ’θ吻oo吻ノ孟αη5 catalyzes the peroxidation reaction with ubiquinol as a
substrate， ZmCytC may accept electrons f士om ubiquinol in addition to cytochrome c． In
the fb㎜er case， the electron trans免r occurs as the order of取pe II NADH
dehydro genase−ubiquinol−ZmCytC． In the latter case， electrons are transfbrred as type II
NADH dehydrogenase−ubiquinol−cytochromeゐ01 complex−cytochrome c552−ZmCytC．
Since there is no cytochrome c oxidase in Z〃70ゐ∫1∫5［Sootsuwan et al．，2007］， it is
thought that ZmC外C plays an impo貢ant role in the fb㎜翫ion of membrane potential at
the cytochrome bcl complex site． Further study is required fbr this speculation．
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Characterization of c乃∫and p5∫． J Biol Chem 1993；268：11779−11784．
Yamada H Takashima g E Konishi K Molecular characterization of the membrane−bound
， ，
，
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274：S53−66．
Y㎜㎜oto N， N欲醜ashi K， Nak㎜ichi T， Yoshino M， T欲ai Y， Touda Y， F㎜bayashi
A，KirOyo S， Dose H， Hasegawa M， Datsenko KA， Nakayashiki T， Tomita M，
W㎜er BL， Mori H， Update on the Keio collection of E5c乃θ7励∫o co1∫
single−gene deletion mutants． Mol Syst Biol 2009；5：335−337．
Yang S， Pelletier DA， Lu TYS， Brown SD， The 2ン駕oη20ηα3加ob／1∫5 regulator吻
contributes to tolerance against multiple lignocellulosic pretreatment inhibitors．
BMC Microbiol 2010；135：1−11．
Yang W， Ni L， Somerville RL，‘Astationary−phase protein of E5c乃θ71c乃’αooll that affbcts
the mode of association between the疏ρrepressor protein and operator−bearing
DNA． Proc Natl Acad Sci USA 1993；90：5796−5800．
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Yoo SH， Yoon EJ， Cha J， Lee HG， Antit㎜or activi取of levan polysaccharides丘om
selected microorganisms． Int J Biol Macromol 2004；34：37−41．
Yura T， Kanemori M， Morita MT， The heat shock response：regulation and fしmction． In：
Storz G， Hermecke H， editors． B acterial stress responses． Washington， D．C． ASM
Press．2000， PP．3−18．
Zagorski N， Imlay J，
The chemistry behind oxidative damage． ASBMB Today 2009；
33−36．
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Ac㎞owledgements
First and fbremost I would like to thank my advisor Prof Dr． Mamoru Yamada．
He has taught me， both consciously and㎜一consciously， how good experimental in
applied molecular bioscience is done． I appreciate all his contributions oftime and ideas，
to make my Ph．D． experience productive and stimulating．
Beside my advisor， I am also thankf叫fbr Prof Dr． Kaz㎜obu Matstushita，
Dr． Tomoyuki Ko saka and Dr． Toshiharu Yakushi fbr their helpfUl discussion， as well as
the collaboration， and Dr． Ponthap Thanonkeo fbr encouraging me to study on this
P「09「am・
Iwould also like to ac㎞owledge the Yamaguchi University ofHcers who pay
high attention and was always ready to help me． I have appreciated their collaboration．
1㎜especially th曲ng my reading co㎜i賃ee members飴r their time， interest，㎝d
help釦l co㎜ents．
Iwould like to thank R勾amangala University of Tec㎞ology Tawan−ok，
Thailand fbr financial support throughout my doctoral course．
My time at Y㎜aguchi Unversity was made erjoyable in large part due to the
many丘iends and groups that became a part of my lifb． I am gratefUl fbr time spent with
all my f士iends， fbr my finished up my degree， and fbr many other people and memories．
Lastly， I would like to thank my family fbr all their love and encouragement fbr
my parents who raised me with a love of science and supported me in all my pursuits．
And most of all fbr my loving， supportive， encouraging， and patient husband Noi and
my lovely son Pang Pang whose faithfUl backup support my heart during the stages of
this Ph．D． is so appreciated． Thank you．
Kamikar Charoensuk
Yamaguchi University
May 2011
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Summ劉ry
Molecul劉r mech劉nism fbr coping with critic劉I he紐t and oxidative
StreSSeS in thermOtOlemnt 2か〃20〃20πα5〃ωわ’1∫5
High−temperahlre色㎜entation tec㎞ology with the㎜otolerant mesophiles is
expected to become one of the economical next−generation魚㎜entation tec㎞ologies，
reduces cooling cost， contamination risk and operation cost in addition to advantages
speci且c fbr ethanol production including higher sacchari且cation and飴㎜entation rates
and continuous ethanol removal． For survival at high temperature， the㎜otolerant
mesophiles are expected to possess a mechanism dif飴rent丘om non−the㎜otolerant
mesophiles． I thus fbcused on molecular mechanisms supporting survival at a critical
high temperahlre（CHT）in E80乃εア∫6伽co1∫and the㎜otolerantみ脚o溺oηα脚o励5 that
can be alive at 47°C and 39°C， respectively． Additionally， one of strategies fbr dealing
with oxidative stress in Z加oゐ∫1∫5 was investigated since oxidative stress is increased as
temperature is elevated．
恥c乃θ7た伽oo1’genome−wide screening with a single−gene㎞ockout library
provided a list of genes indispensable fbr growth at 47°C， called the㎜otolerant genes．
Genes fbr which expression was affbcted by exposure to CHT were identified by DNA
chip analysis． Unexpectedly， the fb㎜er contents did not overlap with the Ia就er except
fbr∂カα1 and 6加α」鴎indicat元ng that a specific set of non。heat shock genes is required fbr
the organism to survive under such a severe condition． More than half of the mutants of
the the㎜otolerant genes were飴und to be sensitive to H202 at 30°C， suggesting th就the
mechanism of the㎜otolerance pa而ally overlaps with th翫of oxidative s廿ess resistance．
Their encoded enzymes or proteins are related to outer membrane organization， DNA
double−strand break repair， tRNA modification， protein quality control， translation
control or cell division． DNA chip analyses of essential genes suggest that many of the
genes encoding ribosomal proteins are down−regulated at CHT． Bioinfb㎜atics analysis
and comparison with the genomic infb㎜ation of other microbes suggest that E． oo1’
possesses several systems fbr survival at CHT． This analysis allows us to speculate that
alipopolysaccharide biosynthesis system fbr outer membrane organization and a
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sulfUr−relay system fbr tRNA modification have been acquired by horizontal gene
transfbr．
An ethanologenic the㎜otolerant Z脚oゐ∫1’5 TISTR 548 strain that was chosen as
athe㎜otolerant s廿a血甲as s呵ected to廿ansposon mutagenesis via transco可ugation
with a mobilizable plasmid harboring the transposable element（Tn10）and growth
experiment at 39°C， CHT fbr the strain． Among about 4，0000f the transco切ugants
obtained，42 mutants that were fbund to be dramatically defbctive in growth at the CHT，
which were selected as the㎜osensitive mutant strains． The insertion site of Tn10within
the genome was then dete㎜ined by the㎜al asymmetric interlaced−（TAIL）PCR
飴llowed by DNA sequencing． As a result，17genes related to the the㎜otolerance have
been identified． Some of these were related to membrane biosynthesis and lipid
metabolism， recombination fbr DNA repair and replication， tRNA modification，
transportation system， which may have a direct or in g direct relation to the
the㎜otolerance mechanism． Interestingly， the results also revealed a pa賞ial
overlapping between genes required fbr the themotolerance and those fbr toIerance to
other stresses． Our findings provide molecular mechanisms underlying a survival of Z
刑oろ’1∫3at CHT which may advantages in production of ethanor and other usefUl
materials at high temperature． ，
Z〃20ゐ’1∫5ZmCytC as a peroxidase bearing three heme c−binding」motif忌was
investigated with∠Zη2の∼∫C constructed． The mutant exhibited filamento亡s shapes and
reduction in growth under a shaking condition at a high temperature compared to the
parental strain and became hypersensitive to exogenous H202． Under the same condition，
the mutation caused increased expression of genes fbr three other antioxidant enzymes．
Peroxidase activity， which was detected in membrane fセactions with ubiquinol−1 as a
substrate but not with reduced horse heart cytochrome o， was almost abolished i11
、4Z〃2のノ∫C． Peroxidase activity waS also detected with NADH as a substrate， which was
significantly inhibited by antimycin A． NADH oxidase activity of、」Zη2（γC was fbund
to be about 80％of that of the parental strain． The results suggest the involvement of
ZmCytC in the aerobic respiratory chain via the cytochromeわ01 complex in addition to
the previously proposed direct interaction with ubiquinol and its contribution to
protection against oxidative stress．
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Summ訊ry
（Jap劉nese）
題目：耐熱性み脚〃20η05〃20ろ伽における限界熱ストレスと酸化ストレスに対す
る対処分子機構（M・lecular mechanism飴r c。ping with critical heat and。xidative stresses in
therlnotolerant 2ン〃zo〃20ηα3〃20Z）∫1∫3）
耐熱性中温菌を用いた高温発酵技術は、高い糖化性や発酵率と連続的エタノ
ー ル除去を含むエタノール生産に関する利点に加えて冷却コスト削減・雑菌混
入抑制・操作コスト削減を可能にする、経済的な次世代発酵技術として注目さ
れている。耐熱性中温菌は、高温下での生存のために非耐熱性中温菌とは異な
った機構をもつことが予想される。本研究では47℃で生育可能なE50乃爾c乃∫oco1∫
と39℃で生育可能な2卿o〃20ηα5〃20b∫1∫5おいて、生育限界温度（CHT）での生存
分子機構に焦点を当てた。さらに、温度上昇に伴って酸化ストレスが増加する
ことから、酸化ストレスに対処するための分子機構についても検討した。
Eco1’のゲノムワイドスクリーニングによって47℃での生育に不可欠な遺
伝子（耐熱性遺伝子）群を同定した。CHTショックによって発現変動する遺伝
子群を同定した。予想に反して、前者は4ηα1と伽oKを除いて後者と重複して
おらず、CHTでは特殊な非熱ショック応答遺伝子セットが要求されることが示
された。耐熱性遺伝子変異株の半分以上が30℃で過酸化水素に感受性であるこ
とが判明し、耐熱性機構は部分的に酸化ストレス耐性機構と重複していること
が示唆された。耐熱性遺伝子は、外膜の形成、DNA二本鎖切断修復、 tRNA修
飾、タンパク質品質管理、転写調節、細胞分裂に関係していた。必須遺伝子の
内、多くのリボソームタンパク質遺伝子がCHTで発現抑制されることが示され
た。生物情報学的解析とゲノム情報比較によって、Eoo1∫が生育限界温度での生
存のためにいくつかのシステムをもつことが示唆された。特に、リボ多糖の生
合成系とtRNA修飾のための硫黄リレー系が水平伝播によって獲得されてきた
と推測された。
エタノール生産性の耐熱性Z脚わ∫1’5TISTR 548株について、トランスポゾン
（Tn10）変異処理によって42個のCHT感受性変異株を取得した。ゲノムへの
Tn10の挿入箇所は、 TAIL PCRとDNAシークエンシングによって決定した。そ
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の結果、17個の耐熱性遺伝子を同定した。これらの遺伝子は、膜の生合成や脂
質代謝、DNA組換え修復と複製、 tRNA修飾、輸送系に関与することが分かっ
た。また本研究によって、耐熱性遺伝子群と他のストレス耐性遺伝子群の間で
部分的に重複することが示唆された。本研究で明らかになったCHTでの生存分
子機構はエタノールや有用物質の高温発酵に活かされると期待される。
Z〃20励3のペルオキシダーゼ（ZmCytC）は3つのヘムc結合モチーフをも
ち、酸化ストレスに対処していると予想される。Z〃卿C欠損変異株は、高温撹
i絆条件下において親株と比較して繊維状形態が顕著となり生育の減少を示し、
過酸化水素に対してより強い感受性を示した。Z撒ッだ欠損変異は、他の3つの
抗酸化酵素遺伝子の発現上昇を引き起こした。ベルオキシダーゼ活性はユビキ
ノールー1やNADHを基質として膜画分で検出できた。 Z〃2（ッ∫C欠損株ではユビキ
ノールー1一ベルオキシダーゼ活1生がほとんど見られなかった。NADH一ベルオキシ
ダーゼ活性はアンチマイシンAによって強く阻害されたが、Z〃πγC欠損株では
NADHオキシダーゼ活性は親株の80％程度であった。この結果は以前に提唱さ
れたユビキノールとの直接的相互関係と酸化ストレスに対する防御への貢献に
加えて、チトクロムゐc1複合体を介した好気的呼吸鎖におけるZmCytCの関与を
示唆する。
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I」ist of publications
1．K紐nnikar Charoensuk， A｝dra Irie， Noppon Lertwattanasaku1， Kaewta Sootsuwan，
Pomthap Thanonkeo and Mamgru Y㎜ada：
Physiological importance of cytochrome c peroxidase in eth3nologenic
thermotolemnt Z卿o脚πα5配o峨5． Journol of Molecular Microbiology and
Biotechnology Vol．20（2011）， No．2pp．70−82（Chapt6r 4）． ’
2．Masayuki Murata， Hiroko F吋imoto， Kaori Nishimura， Kannikar Ch劉roensuk，
Hiroshi Nagamitsu， Satish Raina， Tomoyuki Kosaka， Taku Oshima， Naotake
Ogasawara， Mamoru Yamada：Molecular strategy for survival at a critical high
temper飢ure in Eぎc乃’8ア∫c乃’αco〃Journol of PLρS ONE， in press（Ch訊pter 2）
3． 題目：耐熱性エタノール生産細菌を用いたバイオエタノール生産技術
著者名：山田 守，カニカ・チャロエンスク，入江 陽，ケウタ・スーツワン，
ポンテップ・サノンケオ
学術雑誌名 巻・号・頁：ケミカルエンジニヤリング53：681−686
発表又は受理：発表
発刊又は発刊予定年月：09／2008
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Japan-U舐ted states Reーati。ns i= the Gー。baー C。ntext

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Invitation

Intmduc廿on

ALB Emtia Raporu 27 03 2015

Chapter 7.3-7.4

SAMPLE QUESTION PAPER XII- BIOLOGY FROM JNV

WORKSHEET 6.4-6.6 Section 6.4 – Traits, Genes and Alleles 1 - Mr