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T%e role" o( m!$, an alginate in t%e 'ir!len$e o( P"e!omona" aer!gino"a in
plant"; nematoe" an mi$e
Peter Yorge/3;<;
La!ren$e )= Ra%me>;
Man*Wa% Tan3;?;
#reeri$k M= A!"!&el3;@
Arti$le 5r"t p!&li"%e onlineA B JUL >77C
,OIA 37=372D6E=38D4*>F4C=>773=7>4C7=:
I""!e
Mole$!lar Mi$ro&iolog/
1ol!me 23; I""!e 4; page" 37D8G37BD; Septem&er >773
Aitional In(ormationHS%o+ AllI
Ho+ to Cite A!t%or In(ormation P!&li$ation Hi"tor/
Ho+ to Cite
Yorge/; P=; Ra%me; L= )=; Tan; M=*W= an A!"!&el; #= M= H>773I; T%e role" o( m!$, an
alginate in t%e 'ir!len$e o( P"e!omona" aer!gino"a in plant"; nematoe" an
mi$e= Mole$!lar Mi$ro&iolog/; 23A 37D8G37BD= oiA 37=372D6E=38D4*
>F4C=>773=7>4C7=:
A!t%or In(ormation
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Intro!$tion
Re"!lt"
,i"$!""ion
E:perimental pro$e!re"
A$kno+legement"
Re(eren$e"
We are e:ploiting t%e &roa %o"t range o( t%e %!man opport!ni"ti$ pat%ogen
P"e!omona" aer!gino"a "train PA32 to el!$iate t%e mole$!lar &a"i" o( &a$terial
'ir!len$e in plant"; nematoe"; in"e$t" an mi$e= In t%i" report; +e $%ara$teri9e t%e
role t%at t+o PA32 gene pro!$t"; M!$, an Alg,; pla/ in 'ir!len$e= M!$, i"
ort%ologo!" to t%e E"$%eri$%ia $oli peripla"mi$ protea"e an $%aperone ,egP= ,egP
%omolog!e" are kno+n 'ir!len$e (a$tor" t%at pla/ a prote$ti'e role in "tre""
re"pon"e" in 'ario!" "pe$ie"= Alg, i" an en9/me in'ol'e in t%e &io"/nt%e"i" o( t%e
e:opol/"a$$%arie alginate; +%i$% i" %/perin!$e in m!$, m!tant"= A PA32 m!$,
m!tant +a" "igni5$antl/ impaire in it" a&ilit/ to $a!"e i"ea"e in Ara&iop"i"
t%aliana an mi$e an to kill t%e nematoe Caenor%a&iti" elegan"= Moreo'er; M!$,
+a" (o!n to &e reO!ire (or t%e pro!$tion o( an e:tra$ell!lar to:in in'ol'e in C=
elegan" killing= In $ontra"t; a PA32 alg, m!tant +a" not impaire in 'ir!len$e in
plant"; nematoe" or mi$e= A m!$,alg, o!&le m!tant %a t%e "ame p%enot/pe a"
t%e m!$, "ingle m!tant in t%e plant an nematoe pat%ogene"i" moel"= Ho+e'er;
t%e m!$,alg, o!&le m!tant +a" "/nergi"ti$all/ re!$e in 'ir!len$e in mi$e;
"!gge"ting t%at alginate $an partiall/ $ompen"ate (or t%e lo"" o( M!$, (!n$tion in
mo!"e pat%ogene"i"=
Intro!$tion
Top o( page
A&"tra$t
Intro!$tion
Re"!lt"
,i"$!""ion
E:perimental pro$e!re"
A$kno+legement"
Re(eren$e"
To e:plore $on"er'e me$%ani"m" o( &a$terial pat%ogene"i" an %o"t e(en$e; +e
are !"ing a m!lti%o"t moel "/"tem t%at in'ol'e" t%e %!man opport!ni"ti$ pat%ogen
P"e!omona" aer!gino"a= T%i" moel take" a'antage o( t%e (a$t t%at P= aer!gino"a
"train UCBPP*PA32 HPA32I $a!"e" i"ea"e in plant" HAra&iop"i" t%aliana an
lett!$eL Ra%me et al=; 3FF4L 3FFBI; in"e$t" H)alleria mellonella an ,ro"op%ila
melanoga"terLJaner et al=; >777I an mi$e HRa%me et al=; 3FF4I= PA32 al"o kill" t%e
nematoe Caenor%a&iti" elegan" HMa%aEan*Miklo" et al=; 3FFFL Tan et al=; 3FFFaI=
Pre'io!" +ork (rom o!r la&orator/ %a" "%o+n t%at t%ere i" a remarka&le egree o(
o'erlap &et+een t%e 'ir!len$e (a$tor" reO!ire (or PA32 pat%ogeni$it/ in it" plant
an animal %o"t" HRa%me et al=; 3FF4L 3FFBL Ma%aEan*Miklo" et al=; 3FFFL Tan et al=;
3FFF&I= T%i" permitte t%e relati'el/ rapi ienti5$ation o( no'el P= aer!gino"a
'ir!len$e (a$tor" reO!ire (or ma:im!m pat%ogeni$it/ in mi$e; &/ "$reening PA32
m!tant li&rarie" (or m!tant" t%at are le"" pat%ogeni$ in plant" HRa%me et al=; 3FFBI
or t%at (ail to kill C= elegan" HMa%aEan*Miklo" et al=; 3FFFL Tan et al=; 3FFF&I or )=
mellonella HS= Mi/ata; ,= Lee; J= 1illan!e'a; C= Si(ri; S= Caler+oo an #= A!"!&el;
!np!&li"%e ataI= #!rt%ermore; t%e geneti$ tra$ta&ilit/ o( t%e Ara&iop"i"; C=
elegan" an ,= melanoga"ter %o"t" (a$ilitate" $%ara$teri9ation o( t%e intera$tion
&et+een P= aer!gino"a 'ir!len$e (a$tor" an %o"t target" or e(en$e me$%ani"m"
HMa%aEan*Miklo" et al=; 3FFFI= In t%i" paper; +e take a'antage o( t%e m!lti%o"t
moel "/"tem to $%ara$teri9e t%e role in pat%ogene"i" o( a P= aer!gino"a gene t%at
"%are" "igni5$ant %omolog/ +it% t%e E"$%eri$%ia $oli egP H%trAI gene=
T%e ,egP protein +a" 5r"t ienti5e in E= $oli a" a peripla"mi$ "erine protea"e t%at
egrae" improperl/ (ole or amage protein" an t%at i" reO!ire (or gro+t% at
%ig% temperat!re" HStra!$% an Be$k+it%; 3FCCL Lipin"ka et al=; 3FCFL 3FF7L Stra!$%
et al=; 3FCFI= Heat "%o$k an ot%er "tre""e" in t%e $ell en'elope in!$e e:pre""ion
o( egP 'ia t%e alternate "igma (a$tor RpoE; t%e RpoE reg!lator" R"eAB an t%e
t+o*$omponent "en"orGa$ti'ator pair Cp:RA HConnoll/ et al=; 3FFBL Rai'io an
Sil%a'/; 3FFFI= Re$entl/; E= $oli ,egP %a" al"o &een "%o+n to %a'e $%aperone
a$ti'it/ t%at i" $apa&le o( pro'iing at lea"t "ome prote$tion (rom %eat "tre""
inepenentl/ o( it" proteol/ti$ a$ti'it/ HSpie"" et al=; 3FFFI= In aition to egP; E=
$oli $ontain" t+o egP %omolog!e"; egP H%%oAI an egS H%%oBI HBa"" et al=;
3FFDL Waller an Sa!er; 3FFDI= Alt%o!g% neit%er o( t%e"e gene" i" reO!ire (or
gro+t% at %ig% temperat!re; $on"tit!ti'e e:pre""ion o( egP H&!t not egSI
$omplement" t%e ina&ilit/ o( a egP m!tant to gro+ at %ig% temperat!re HWaller
an Sa!er; 3FFDI= egP %omolog!e" %a'e &een ienti5e in n!mero!" )ram*
negati'e &a$teria a" +ell a" in )ram*po"iti'e &a$teria; $/ano&a$teria; /ea"t; plant"
an %!man" HPallen an Wren; 3FFBL It9%aki et al=; 3FFCI=
In aition to t%eir prote$ti'e role" in "tre"" re"pon"e"; ,egP %omolog!e" %a'e al"o
&een "%o+n to pla/ a role in pat%ogene"i" in "e'eral animal pat%ogen"= in$l!ing
Salmonella t/p%im!ri!m; Yer"inia entero$oliti$a an Br!$ella a&ort!" HJo%n"on et al=;
3FF3L Ba!mler et al=; 3FF2L El9er et al=; 3FF2L 3FFDL Roop et al=; 3FF2L Tat!m et al=;
3FF2L Li et al=; 3FFDL Yamamoto et al=; 3FFDL 3FFBI= S= t/p%im!ri!m egP m!tant";
(or e:ample; e:%i&it re!$e 'ir!len$e in mi$e HJo%n"on et al=; 3FF3I; re!$e
"!r'i'al in ma$rop%age" HBa!mler et al=; 3FF2I an en%an$e "en"iti'it/ to
o:iati'e killing in 'itro HJo%n"on et al=; 3FF3I; $on"i"tent +it% t%e %/pot%e"i9e role
o( ,egP in prote$ting S= t/p%im!ri!m (rom t%e eleterio!" eQe$t" o( t%e
ma$rop%age o:iati'e &!r"t HJo%n"on et al=; 3FF3I= Moreo'er; in +ork (rom o!r
la&orator/; a ,egP %omolog!e +a" ienti5e in t%e plant pat%ogen P"e!omona"
"/ringae p'= ma$!li$ola "train ES28>D in a "$reen (or P= "/ringae m!tant" t%at
e:%i&ite re!$e 'ir!len$e in Ara&iop"i" HSte'en"; 3FFCI= Intere"tingl/; in
re"pon"e to pat%ogen atta$k; plant"; like animal"; al"o e:%i&it a pat%ogen*in!$e
o:iati'e &!r"t; +%i$% appear" to a$ti'ate a 'ariet/ o( e(en$e re"pon"e" in plant"
HAl'are9 et al=; 3FFCI=
In t%i" report; +e e"$ri&e t%e $%ara$teri9ation o( a P= aer!gino"a PA32 egP
%omolog!e t%at i" tig%tl/ $l!"tere +it% "e'eral ot%er gene" in'ol'e in reg!lation
o( t%e "/nt%e"i" o( t%e e:opol/"a$$%arie alginate H)o'an an ,ereti$; 3FFDI= T%e
PA32 egP gene $orre"pon" to t%e P= aer!gino"a "train PAO3 m!$, gene;
m!tation" in +%i$% re"!lt in t%e (ormation o( m!$oi $olonie" a" a re"!lt o( t%e
o'erpro!$tion o( alginate HBo!$%er et al=; 3FFDI= Be$a!"e alginate %a" &een
impli$ate a" a ke/ 'ir!len$e (a$tor in $%roni$ P= aer!gino"a in(e$tion" in t%e l!ng"
o( $/"ti$ 5&ro"i" patient" an &e$a!"e ,egP %omolog!e" in ot%er "pe$ie" are
important 'ir!len$e (a$tor"; it +a" o( intere"t to etermine +%et%er P= aer!gino"a
PA32 M!$, i" a 'ir!len$e (a$tor in plant; nematoe; in"e$t or mo!"e pat%ogene"i"
an; i( "o; +%et%er t%i" i" relate to it" role a" a reg!lator o( alginate &io"/nt%e"i"=
O!r re"!lt"; e"$ri&e in t%i" paper an in a re$ent p!&li$ation HJaner et al=; >777I;
ini$ate t%at M!$, i" an important 'ir!len$e (a$tor in plant; nematoe; in"e$t an
mammalian %o"t" an t%at M!$, i" reO!ire (or t%e pro!$tion o( an e:tra$ell!lar
to:in in'ol'e in C= elegan" killing= On t%e ot%er %an; t%e P= aer!gino"a PA32 Alg,
protein an; &/ in(eren$e; it" &io"/nt%eti$ en*pro!$t alginate pla/ a limite role; i(
an/; in plant; nematoe an mammalian %o"t"; e:$ept in a m!$, m!tant
&a$kgro!n in +%i$% t%e/ are ne$e""ar/ (or pat%ogene"i" in a mo!"e t%ermal &!rn
inE!r/ moel= In general; o!r re"!lt" emon"trate t%e !tilit/ o( t%e m!lti%o"t
pat%ogene"i" moel in $%ara$teri9ing t%e "pe$i5$ role" o( P= aer!gino"a 'ir!len$e
(a$tor" in pat%ogene"i"=
Re"!lt"
Top o( page
A&"tra$t
Intro!$tion
Re"!lt"
,i"$!""ion
E:perimental pro$e!re"
A$kno+legement"
Re(eren$e"
Cloning an ,NA "eO!en$e anal/"i" o( a P= aer!gino"a PA32 egP %omolog!e
A P= aer!gino"a PA32 egP %omolog!e +a" ienti5e &/ "$reening a $o"mi li&rar/
o( PA32 $%romo"omal ,NA +it% a pro&e eri'e (rom a P= "/ringae egP*like gene
H"ee E:perimental pro$e!re" (or etail"I= Re"tri$tion mapping; "!&$loning; ,NA
&lot anal/"i" an ,NA "eO!en$e anal/"i" o( a region o( t%e PA32 genome t%at
%/&rii9e to t%e P= "/ringae egP pro&e ienti5e a PA32 egP %omolog!e lo$ate
immeiatel/ o+n"tream o( a gro!p o( gene" in'ol'e in t%e reg!lation o( alginate
&io"/nt%e"i"; algU HalgTI; m!$A; m!$B HalgNI an m!$C HalgMI H)ol&erg et al=;
3FF8L Martin et al=; 3FF8L ,ereti$ et al=; 3FF2L ,e1rie" an O%man; 3FF2L Martin
et al=; 3FF2L Her"%&erger et al=; 3FF4L S$%!rr et al=; 3FFDL Rie et al=; 3FFDL Mat%ee
et al=; 3FFBI= T%e "ame P= aer!gino"a egP %omolog!e +a" ienti5e inepenentl/
in P= aer!gino"a "train PAO3 an e"ignate m!$, HBo!$%er et al=; 3FFDI= It appear"
likel/ t%at t%e egP6m!$, region" o( t%e PA32 an PAO3 genome" %a'e t%e "ame
organi9ation= Pol/mera"e $%ain rea$tion HPCRI primer" $orre"poning to t%e PA32
egP6m!$, "eO!en$e an to t%e p!&li"%e PAO3 m!$B "eO!en$e ampli5e a
prei$te 4C7 &p pro!$t (rom PA32 $%romo"omal ,NA; $on"i"tent +it% t%e "ame
gene arrangement in PA32 an PAO3 Hata not "%o+nI=
,NA "eO!en$e anal/"i" o( t%e PA32 egP open reaing (rame HOR#I re'eale a
prei$te protein pro!$t t%at +a" ienti$al in lengt% H2B2 amino a$i"I &!t t%at
$ontaine (o!r $on"er'ati'e amino a$i iQeren$e" $ompare +it% t%e PAO3 m!$,
pro!$t Hpo"ition PA32APAO3L 38B1AIL >>4 EAPL 8D8 NASL 223 IA1I HBo!$%er et al=;
3FFDI= O!t o( 3434 &p o( PA32 ,NA "eO!en$e $ompri"ing m!$, an Sanking
region"; t%ere +ere 3F n!$leotie iQeren$e" &et+een t%e PAO3 an PA32
"eO!en$e"= A$$oringl/; +e %a'e e"ignate t%e PA32 egP %omolog!e m!$,=
T%e"e "eO!en$e ata %a'e &een "!&mitte to t%e ,,BJ6EMBL6)enBank ata&a"e"
!ner a$$e""ion n!m&er A#828FB8=
Con"tr!$tion o( a PA32 m!$, m!tant
To i"r!pt t%e PA32 m!$, gene; a gene $a""ette en$oing gentami$in re"i"tan$e;
aa$C3; +a" $lone into a R%oI "ite lo$ate in t%e N*terminal en o( t%e PA32 m!$,
OR#= T%i" m!$,AAaa$C3 $on"tr!$t +a" t%en !"e to repla$e t%e +il*t/pe $op/ o(
m!$, in t%e PA32 genome &/ marker e:$%ange !"ing t%e po"iti'e "ele$tion "!i$ie
'e$tor pC1,22> H,onnen&erg an Naper; 3FF3I= T%e "tr!$t!re o( t%e m!$,AAaa$C3
i"r!ption m!tation +a" $on5rme &/ ,NA &lot anal/"i" Hata not "%o+nI= T%e
logarit%mi$ gro+t% rate o( t%e PA32 m!$, m!tant +a" t%e "ame a" +il*t/pe PA32
in &ot% ri$% HLB or NBI an minimal HMFI meia= Ho+e'er; t%e m!$, m!tant o(ten
e:%i&ite a 3 or > % longer lag &e(ore gro+t% $ommen$e $ompare +it% +il*t/pe
PA32 +%en a "at!rate $!lt!re +a" "!&$!lt!re into ri$% or minimal meia= W%erea"
+il*t/pe PA32 gre+ +ell on "oli meia at 2>TC; t%e PA32 m!$, m!tant e:%i&ite
little or no gro+t%; "imilar to E= $oli egP m!tant" HStra!$% et al=; 3FCFL Lipin"ka
et al=; 3FF7I an t%e P= aer!gino"a PAO3 m!$, m!tant HBo!$%er et al=; 3FFDI= T%e
PA32 m!$, m!tant (orme m!$oi $olonie" a(ter in$!&ation on LB; NB or PIA agar
meia at 8BTC "imilar to t%e PAO3 m!$, m!tant on PIA HBo!$%er et al=; 3FFDI=
T%e PA32 m!$, gene $omplement" PA32 m!$, an E= $oli egP m!tant"
A >=7 k&p E$oRIGSalI (ragment $ontaining t%e PA32 m!$, gene e:pre""e (rom t%e
E= $oli la$U promoter in pla"mi pPY>>3 $omplemente &ot% t%e m!$oi an
temperat!re*"en"iti'e p%enot/pe" o( t%e PA32 m!$, "train H#ig= 3AL ata not
"%o+nI= A "eparate $omplementation e:periment +a" $arrie o!t +it% a 4=8 k&p
(ragment (rom PA32 t%at $ontaine t%e entire algUm!$ABC, $l!"ter= T%i" (ragment;
$arrie on pla"mi pPY>>>; +a" integrate into t%e $%romo"ome o( t%e PA32 m!$,
"train &/ %omologo!" re$om&ination !p"tream o( t%e m!$,AAaa$C3 in"ertion;
lea'ing an/ po""i&le polar eQe$t" o( t%e m!$, in"ertion m!tation inta$t= T%i"
integrant %a t%e "ame p%enot/pe a" +il*t/pe PA32 +it% re"pe$t to temperat!re
"en"iti'it/ an m!$oi/ Hata not "%o+nI= T%e"e re"!lt" "%o+ t%at t%e p%enot/pe"
a""o$iate +it% t%e in"ertion in m!$, re"!lte (rom t%e lo"" o( m!$, e:pre""ion
rat%er t%an an !nlinke m!tation or a polar eQe$t on a o+n"tream gene= In
aition; pla"mi pPY>>3 $ontaining t%e PA32 m!$, gene !ner la$U promoter
$ontrol partiall/ $omplemente t%e temperat!re*"en"iti'e p%enot/pe o( t%e E= $oli
egP23 m!tant H#ig= 3BL Stra!$% et al=; 3FCFI; ini$ating t%at P= aer!gino"a M!$, i"
mo"t pro&a&l/ targete to t%e peripla"m like E= $oli ,egP an %a" at lea"t "ome
o'erlapping "!&"trate "pe$i5$it/ or (!n$tion=
#ig!re 3= T%e PA32 m!$, gene $arrie on pPY>>3 $omplement" t%e m!$oi/ o( t%e
PA32 m!$, m!tant an t%e temperat!re "en"iti'it/ o( an E= $oli egP m!tant=
A= P= aer!gino"a PA32 "train" gro+n on PIA agar at 8BTC (or > a/"= 3; Wil*t/peL >;
m!$,L 8; m!$, pUCP3CL 2; m!$, pPY>>3=
B= E= $oli "train" gro+n on LB agar at 2>TC (or >2 %= 3; Wil*t/pe HNS>B>IL >; egP23
HNS2B2IL 8; egP23 pUCP3CL 2; egP23 pPY>>3=
,o+nloa 5g!re to Po+erPoint
Con"tr!$tion o( a PA32 alg, internal in (rame eletion
A" e"$ri&e &elo+; t%e PA32 m!$, m!tant i" "igni5$antl/ le"" pat%ogeni$ in plant;
nematoe an mo!"e pat%ogene"i" moel"= Ho+e'er; t%e m!$oi p%enot/pe o( t%e
m!$, m!tant rai"e t%e $on$ern t%at t%e o'erpro!$tion o( alginate per "e ma/ &e
t%e $a!"e o( re!$e 'ir!len$e rat%er t%an t%e lo"" o( t%e M!$, protea"e or
$%aperone a$ti'it/= To are"" t%i" $on$ern an to etermine t%e role o( alginate in
'ir!len$e in o!r pat%ogene"i" moel"; +e $on"tr!$te a PA32 alg, m!tant an a
m!$,alg, o!&le m!tant=
T%e alg, gene; t%e 5r"t gene in a large operon $ontaining gene" reO!ire (or
alginate &io"/nt%e"i"; en$oe" ),P*manno"e e%/rogena"e; t%e 5r"t $ommitte
"tep in alginate &io"/nt%e"i" H)o'an an ,ereti$; 3FFDI= )i'en t%at it i" !nkno+n
+%et%er all t%e gene" in t%i" operon are ei$ate "olel/ to alginate &io"/nt%e"i";
an internal in (rame eletion in t%e PA32 alg, gene +a" $on"tr!$te to pre'ent an/
polar eQe$t" on o+n"tream gene"= A" e"$ri&e in E:perimental pro$e!re"; a
PCR*&a"e approa$% +a" !"e to generate a eletion in t%e PA32 alg, gene t%at
retain" t%e 5r"t "i: an la"t 8D $oon" o( t%e gene= T%e elete alg, gene +a" t%en
!"e to repla$e t%e +il*t/pe $op/ o( alg, in PA32 an in PA32 m!$,= A" e:pe$te;
t%e m!$,alg, o!&le m!tant +a" no longer m!$oi on PIA plate"; $on5rming t%at
t%e m!$oi p%enot/pe o( t%e m!$, m!tant re"!lte (rom t%e o'erpro!$tion o(
alginate= In aition; &e$a!"e pla"mi pPY>>D; +%i$% $ontain" onl/ a +il*t/pe $op/
o( alg,; re"tore m!$oi/ in t%e m!$,alg, m!tant &a$kgro!n; +e $on$l!e t%at
t%e alg, eletion oe" not %a'e a polar eQe$t on o+n"tream alginate &io"/nt%eti$
gene" Hata not "%o+nI=
In t%e "e$tion" t%at (ollo+; t%e PA32 m!$,; alg, an m!$,alg, o!&le m!tant"
+ere te"te (or a 'ariet/ o( p%enot/pe" in $ompari"on +it% +il*t/pe PA32=
Lo"" o( M!$, (!n$tion $on(er" en%an$e temperat!re an o:iati'e "tre""
"en"iti'it/; +%erea" lo"" o( Alg, %a" no ete$ta&le impa$t
To e:ten o!r earlier o&"er'ation t%at PA32 m!$, e:%i&ite temperat!re "en"iti'it/;
+e a""e""e +%et%er t%e alg, m!tation %a an/ impa$t on t%i" p%enot/pe= #re"%
o'ernig%t $!lt!re" o( +il*t/pe PA32 or t%e alg, m!tant "%o+e no lo"" o( 'ia&ilit/
a(ter tran"(er to 24TC (or 2 %; +%erea" t%e PA32 m!$, m!tant an t%e m!$,alg,
o!&le m!tant &ot% e:%i&ite an appro:imate >7*(ol e$rea"e in 'ia&ilit/ Hata not
"%o+nI=
In$rea"e "en"iti'it/ to o:iati'e "tre"" in 'itro %a" al"o &een reporte (or a 'ariet/
o( egP*like m!tant"; in$l!ing PAO3 m!$, HBo!$%er et al=; 3FFDI= In aition;
alginate %a" &een %/pot%e"i9e to pro'ie prote$tion again"t o:iati'e "tre"" an
%a" &een "%o+n to "$a'enge %/po$%lorite an rea$ti'e o:/gen "pe$ie" generate
&/ ma$rop%age" an ne!trop%il" HLearn et al=; 3FCBL Simp"on et al=; 3FCFI=
Sen"iti'it/ to o:iati'e "tre"" +a" te"te a" e"$ri&e in E:perimental pro$e!re"
!"ing 4V H>O>*impregnate 5lter i"k" pla$e on top o( agaro"e o'erla/"
$ontaining "!"pen"ion" o( t%e m!tant an +il*t/pe "train"= To a'oi $ompli$ation"
a" a re"!lt o( t%e temperat!re "en"iti'it/ o( t%e m!$, m!tant; lo+*melting*point
agaro"e +a" eO!ili&rate at 8BTC &e(ore t%e aition o( t%e PA32 "train" an
po!ring t%e o'erla/"= #ig!re > "%o+" t%at; !ner t%e $onition" te"te; t%e m!$,
an m!$,alg, m!tant" &ot% e:%i&ite a $ompara&le le'el o( en%an$e "en"iti'it/
to H>O>; +%erea" t%e alg, m!tant +a" "imilar to +il t/pe=
#ig!re >= H/rogen pero:ie "en"iti'it/ o( +il*t/pe PA32 'er"!" m!$,; alg, an
m!$,alg, m!tant"= #or ea$% "train; 4 Wl o( a 7=> AD77 "!"pen"ion Hin 37 mM
MgSO2I +a" ae mlX3 3=4V lo+*melting*point agaro"e at 8BTC; +%i$% +a" !"e
to po!r 4 ml o'erla/" on LB plate"= An aliO!ot o( 37 Wl o( 4V H>O> +a" ae to a
"ingle 5lter i"k on ea$% plate= Plate" +ere in$!&ate o'ernig%t at 8BTC; an t%e
iameter" o( t%e 9one" o( in%i&ition +ere mea"!re= T%e"e ata repre"ent t%e mean
an "tanar e'iation (rom a total o( (o!r plate" per "train= ,iQerent letter"
ini$ate "tati"ti$all/ "igni5$ant iQeren$e" &et+een m!tant" H#i"%erJ" lea"t
"igni5$ant iQeren$e" te"t; Z 7=74I=
,o+nloa 5g!re to Po+erPoint
T%e PA32 m!$, an m!$,alg, m!tant" e:%i&it e$rea"e gro+t% in Ara&iop"i";
+%erea" t%e alg, m!tant i" !nimpaire
A" ill!"trate in #ig= 8; a(ter in5ltration o( [372 $(! $mX> lea( area into an
Ara&iop"i" lea(; t%e gro+t% o( t%e PA32 m!$, an m!$,alg, m!tant" +a"
"igni5$antl/ impaire $ompare +it% +il*t/pe PA32 an t%e alg, m!tant= T%e
gro+t% o( t%e PA32 alg, an m!$,alg, m!tant"; %o+e'er; +a" not "igni5$antl/
iQerent (rom t%at o( t%e +il*t/pe an m!$, "train" re"pe$ti'el/=
#ig!re 8= )ro+t% o( +il*t/pe PA32 'er"!" m!$,; alg, an m!$,alg, m!tant" in
Ara&iop"i"= One %!nre*(ol il!tion" o( 7=> AD77 &a$terial "!"pen"ion" Hin 37 mM
MgSO2I +ere in5ltrate into ini'i!al lea'e" o( 4* to D*+eek*ol plant" an
in$!&ate in gro+t% $%am&er" at >FTC= )ro+t% +a" etermine a" $%ange in $(!
per !nit area o( lea( Ha/ 8 "ample" min!" a/ 7 "ample"I= T%e"e ata repre"ent t%e
mean an "tanar error o( t%e mean (rom a total o( 3FG>3 "ample" Hinepenent
lea( "ample"I per m!tant $om&ine (rom 5'e inepenent e:periment"= ,iQerent
letter" ini$ate "tati"ti$all/ "igni5$ant iQeren$e" &et+een m!tant" H#i"%erJ" lea"t
"igni5$ant iQeren$e" te"t; Z 7=74I=
,o+nloa 5g!re to Po+erPoint
T%e 'ir!len$e o( t%e m!$,alg, m!tant i" "/nergi"ti$all/ re!$e in a mo!"e
t%ermal inE!r/ moel
Ta&le 3 "%o+" t%at t%e PA32 m!$, m!tant +a" le"" 'ir!lent in a mo!"e t%ermal
inE!r/ moel $ompare +it% t%e +il t/pe= Pla"mi pPY>>> HalgUm!$ABC,I
$omplemente t%e 'ir!len$e e(e$t o( t%e m!$, m!tant +%en it +a" integrate
!p"tream o( t%e m!$,AAaa$C3 in"ertion; emon"trating t%at t%e 'ir!len$e e(e$t in
t%e m!$, m!tant i" not attri&!ta&le to an !nlinke m!tation or a polar eQe$t o( t%e
in"ertion= In $ontra"t to t%e m!$, m!tant; t%e PA32 alg, m!tant e:%i&ite 'ir!len$e
"imilar to +il t/pe= Intere"tingl/; t%e PA32 m!$,alg, o!&le m!tant +a"
ramati$all/ le"" 'ir!lent t%an t%e m!$, "ingle m!tant; a re"!lt t%at +a"
!ne:pe$te gi'en t%e minimal or a&"ent eQe$t o( t%e alg, m!tation alone= T%e
p%enot/pe o( t%e o!&le m!tant emon"trate" t%at t%e impaire 'ir!len$e o( t%e
m!$, m!tant i" not $a!"e "impl/ &/ t%e o'erpro!$tion o( alginate= In (a$t; t%e"e
re"!lt" "!gge"t t%at alginate ma/ &e partiall/ $ompen"ating (or t%e lo"" o( M!$,
(!n$tion in 'i'o=
Ta&le 3= T%e m!$,alg, m!tant i" a'ir!lent in a mo!"e t%ermal inE!r/ moel=
Strain
No= o( "epti$
mi$e6total
Mi$e t%at e'elope "ep"i" HV
mortalit/I
Si:*+eek*ol male ANR6J mi$e +ere "!&Ee$te to a 4V total "!r(a$e &o/ area
t%ermal inE!r/ on t%eir a&ominal "kin a" e"$ri&e in E:perimental pro$e!re"=
Immeiatel/ a(ter t%e &!rn; mi$e +ere inEe$te +it% 4 \ 374 PA32 $ell"; an t%e
n!m&er o( animal" t%at e'elope "ep"i" +a" monitore ea$% a/ (or 37 a/"=
m!$, M integrate algUm!$ABC, $ontain" t%e pla"mi pPY>>> integrate into t%e
$%romo"ome !p"tream o( t%e m!$,AAaa$C3 in"ertion= T%e #i"%er e:a$t 'al!e"
H'er"!" +il t/peIA m!$, Z 7=734L m!$, M algUm!$ABC,Z 7=27L alg, Z 7=84L
m!$,alg, Z 7=777777= Wit% t%e Bon(erroni $orre$tion (or t%e $ompari"on o( (o!r
te"t gro!p" +it% t%e +il*t/pe $ontrol; a P*'al!e eO!i'alent to 7=74 Hin a t+o*+a/
$ompari"onI i" 7=73>4= U"ing t%i" met%o; t%e m!$, m!tant (all" near to &!t
"lig%tl/ a&o'e t%e 7=73>4 le'el o( "igni5$an$e= T%e m!$,alg, o!&le m!tant i"
$learl/ "igni5$antl/ iQerent=
Ta&le 3= T%e m!$,alg, m!tant i" a'ir!lent in a mo!"e t%ermal inE!r/ moel=
Strain
No= o( "epti$
mi$e6total
Mi$e t%at e'elope "ep"i" HV
mortalit/I
PA32 3263D CCV
m!$, C63B 2BV
m!$, M integrate
algUm!$ABC,
3263D CCV
alg, 3463C C8V
m!$,alg, 763C 7V
T%e m!$, gene pro!$t i" e""ential (or (!ll 'ir!len$e in C= elegan"; +%erea" alg,
pla/" no role
PA32 kill" C= elegan" in a mei!m*epenent manner +%en it i" (e to C= elegan" a"
a "ole "o!r$e o( (oo HMa%aEan*Miklo" et al=; 3FFFL Tan et al=; 3FFFaI= On lo+*
o"molarit/ HminimalI mei!m; appro:imatel/ %al( t%e +orm" are kille &/ 47 % H"lo+
killingI; +%erea" on %ig% o"molarit/ mei!m; at lea"t 47V killing o$$!r" &/ 2 % H(a"t
killingI= Intere"tingl/; "lo+ an (a"t killing appear to &e me$%ani"ti$all/ i"tin$t=
Slo+ killing reO!ire" ire$t $onta$t &et+een li'e &a$teria an +orm" an i"
$orrelate +it% t%e a$$!m!lation o( large n!m&er" o( inta$t PA32 in t%e l!men o(
t%e aQe$te +orm"= A$$!m!lation o( inta$t &a$teria in t%e l!men i" not o&"er'e
+%en C= elegan" i" (e E= $oli= In $ontra"t to "lo+ killing; (a"t killing i" meiate &/
e:tra$ell!lar "mall*mole$!lar*+eig%t to:in" an oe" not reO!ire ire$t $onta$t
&et+een +orm" an li'e &a$teria=
A" "%o+n in #ig= 2A an B; t%e PA32 m!$, m!tant +a" impaire in killing C= elegan"
in &ot% t%e (a"t killing an t%e "lo+ killing a""a/"= In $ontra"t; t%e alg, an
m!$,alg, m!tant" +ere ini"ting!i"%a&le (rom t%e +il t/pe an t%e m!$, m!tant
re"pe$ti'el/ H#ig= 2A an BI; "!gge"ting t%at alg, %a" no eQe$t on 'ir!len$e in
eit%er t%e (a"t or t%e "lo+ killing a""a/"= T%e e:tene time reO!ire (or killing in
t%e "lo+ killing a""a/" H#ig= 2BI pre"ent" t+o e:perimental pro&lem"= #ir"t; t%e rate
at +%i$% egg" %at$% internall/ in %ermap%roite +orm" i" en%an$e +%en +orm"
are (eeing on PA32 $ompare +it% E= $oli= Be$a!"e internal %at$%ing o( egg" kill"
t%e parent; it $an &e i]$!lt to etermine a$$!ratel/ t%e amo!nt o( PA32*meiate
killing= Se$onl/; &e$a!"e PA32 oe" not kill t%e earl/ lar'al "tage"; rapil/
e'eloping oQ"pring o( t%e original L2 H%ermap%roiteI +orm" are i]$!lt to
i"ting!i"% (rom t%eir parent"; on$e again making it i]$!lt to etermine an
a$$!rate rate o( killing= To o'er$ome t%e"e e:perimental i]$!ltie" a""o$iate +it%
t%e pro!$tion o( progen/; a male "terile m!tant H(er*3LA$%an9ar an War; 3FFBI
+a" "!&"tit!te (or +il*t/pe N> +orm"= A" "%o+n in #ig= 2C; t%e PA32 m!$, m!tant
e:%i&ite a "imilar e(e$t in it" a&ilit/ to kill (er*3 +orm" a" in it" a&ilit/ to kill +il*
t/pe +orm"= #ig!re 2C al"o "%o+" t%at t%e integrate pla"mi pPY>>> H$arr/ing
algUm!$ABC,I $omplement" t%e m!$, m!tant to (!ll 'ir!len$e in t%e "lo+ killing
a""a/; emon"trating t%at t%e re!$e 'ir!len$e p%enot/pe o( t%e m!$, m!tant i"
not $a!"e &/ an !nlinke m!tation or a polar eQe$t o( t%e m!$, in"ertion= Pla"mi
pPY>>> al"o (!ll/ $omplemente t%e m!$, m!tation in t%e (a"t killing a""a/ Hata
not "%o+nI=
#ig!re 2= Nilling o( C= elegan" &/ m!$,; alg, an m!$,alg, m!tant" $ompare +it%
+il*t/pe PA32= T%irt/ to 27 L2*"tage +orm" +ere pla$e on la+n" o( t%e ini$ate
PA32 "train" gro+n on P)S or N) agar; in$!&ate at >4TC an "$ore (or li'e6ea
+orm" at t%e ini$ate time"= ,ata repre"ent t%e mean ^ S, o( t%ree repli$ate"=
A= #a"t killing $onition" H%ig%*o"molarit/ P)S mei!mI +it% +il*t/pe N> +orm"=
B= Slo+ killing $onition" Hlo+*o"molarit/ N) mei!mI +it% +il*t/pe N> +orm"=
C= Lo+*o"molarit/ "lo+ killing $onition" +it% $onitional male "terile (er*3 +orm"
H"ee E:perimental pro$e!re" (or etail"I= Strain"A +il t/pe H"oli "O!areIL m!$,
H"oli $ir$leIL alg, Hopen "O!areIL m!$,alg, Hopen $ir$leIL m!$, M algUm!$ABC,
Hopen triangleI=
,o+nloa 5g!re to Po+erPoint
M!$, i" reO!ire (or t%e pro!$tion o( an e:tra$ell!lar 'ir!len$e (a$tor t%at i" not
p/o$/anin
Be$a!"e o!r la&orator/ %a "%o+n pre'io!"l/ t%at (a"t killing i" meiate &/ lo+*
mole$!lar*+eig%t "e$rete to:in"; t%e (a$t t%at t%e PA32 m!$, m!tant i"
ramati$all/ re!$e in it" a&ilit/ to meiate (a"t killing o( C= elegan" "!gge"t" t%at
it i" impaire in t%e pro!$tion or e:port o( an e:tra$ell!lar 'ir!len$e (a$tor or to:in=
T%!"; in aition to it" role" in prote$ting again"t en'ironmental "tre""; M!$, ma/
pla/ a role in t%e pro!$tion o( 'ir!len$e*relate to:in"=
PA32*meiate (a"t killing o( C= elegan" $an o$$!r +it%o!t $onta$t +it% li'e &a$teria
&/ gro+ing a la+n o( PA32 on a 7=24 Wm 5lter pla$e on P)S agar an t%en
remo'ing t%e 5lter (rom t%e agar "!r(a$e HMa%aEan*Miklo" et al=; 3FFFI= W%en L2*
"tage C= elegan" are pla$e on t%i" _$onitione` agar mei!m; t%e +orm" ie +it%
"imilar kineti$" to C= elegan" t%at are in ire$t $onta$t +it% a la+n o( PA32=
Con"i"tent +it% t%e re"!lt" o&taine +it% t%e (a"t killing a""a/ e"$ri&e a&o'e; t%e
PA32 m!$, "train e:%i&ite ramati$all/ re!$e killing $ompare +it% +il*t/pe
PA32 !"ing t%i" 5lter a""a/ HS= Ma%aEan*Miklo"; per"onal $omm!ni$ationI=
One $la"" o( P= aer!gino"a e:tra$ell!lar (a$tor" reO!ire (or (a"t killing are
p%ena9ine"; tri$/$li$ "e$onar/ meta&olite" pro!$e &/ a 'ariet/ o( p"e!omona"
HT!rner an Me""enger; 3FCDI= Alt%o!g% p%ena9ine" are reO!ire; t%e/ are not
"!]$ient (or (a"t killing o( C= elegan"= PA32 m!tant" impaire in t%e C= elegan" (a"t
killing a""a/ (all into at lea"t t+o $la""e"A m!tant" t%at pro!$e re!$e le'el" o(
t%e p%ena9ine p/o$/anin H$la"" II an m!tant" t%at "/nt%e"i9e +il*t/pe le'el" o(
p/o$/anin H$la"" III= T%e m!$,; alg, an m!$,alg, m!tant" "%o+e no re!$tion in
p/o$/anin pro!$tion in &rot% $!lt!re $ompare +it% +il*t/pe PA32; e:%i&iting t%e
(ollo+ing relati'e le'el" o( pro!$tionA +il t/pe; 377V HS, ^3BIL m!$,; 38DV
H^ >7IL alg,; 37FV H^ 2>IL m!$,alg,; >>3V H^ 3FI= S!rpri"ingl/; t%e m!$,alg,
o!&le m!tant repro!$i&l/ pro!$e ele'ate le'el" o( p/o$/anin= It i" !nlikel/ t%at
t%e m!$, m!tant "/nt%e"i9e re!$e le'el" o( p/o$/anin +%en gro+n on P)S agar
in t%e C= elegan" (a"t killing a""a/= P/o$/anin "tain" t%e agar a $%ara$teri"ti$ &l!e*
green or &ro+n $olo!r epening on t%e $ompo"ition o( t%e mei!m; an no $%ange
in $oloration +a" o&"er'e +it% t%e m!$, m!tant in $ompari"on +it% t%e +il t/pe=
Ma%aEan*Miklo" et al= H3FFFI al"o $%ara$teri9e t%e me$%ani"m &/ +%i$%
p%ena9ine" Hp/o$/aninI kill C= elegan" &/ emon"trating t%at t%e C= elegan" P*
gl/$oprotein HP)PI m!tant aNL387 pgp*3Hpk3BIL pgp*8Hpk3CIb e:%i&ite en%an$e
"!"$epti&ilit/ to PA32*meiate (a"t killing $ompare +it% +il*t/pe nematoe"= In
general; P)P" are mem&rane protein" t%at %a'e &een impli$ate a" $omponent" o(
a$ti'e ec!: p!mp" o( a i'er"e range o( e:ogeno!" to:i$ $ompo!n" HHiggin";
3FF>L S$%inkel et al=; 3FF2L Broek" et al=; 3FF4L Higgin"; 3FF4I= Ma%aEan*Miklo"
et al= H3FFFI (o!n t%at t%e en%an$e "!"$epti&ilit/ o( t%e pgp m!tant +orm" +a"
$orrelate +it% p/o$/anin pro!$tion= T%at i"; on %ig% o"molarit/ meia; t%e pgp
m!tant nematoe" "!ppre""e t%e p%enot/pe o( $la"" II PA32 (a"t killing m!tant"
t%at "till "/nt%e"i9e normal le'el" o( p/o$/anin; &!t i not "!ppre"" t%e
p%enot/pe o( $la"" I PA32 (a"t killing m!tant" t%at "/nt%e"i9e re!$e le'el" o(
p/o$/anin= T%i" "!gge"te t%at p/o$/anin Hor ot%er relate p%ena9ine"I +a" a
"!&"trate o( t%e P)P*3 an6or P)P*8 ec!: p!mp"=
Be$a!"e t%e PA32 m!$, m!tant pro!$e at lea"t +il*t/pe le'el" o( p/o$/anin in
&rot% $!lt!re"; +e rea"one t%at pgp*3 pgp*8 m!tant nematoe" "%o!l "!ppre""
t%e 'ir!len$e e(e$t o( t%e m!$, m!tant= Inee; #ig= 4 "%o+" t%at; on %ig%*
o"molarit/ meia; t%e pgp*3 pgp*8 nematoe" +ere almo"t a" "!"$epti&le to killing
&/ t%e m!$, m!tant a" t%e/ +ere to +il*t/pe PA32; $on"i"tent +it% t%e $on$l!"ion
t%at m!$, i" not impaire in p/o$/anin pro!$tion on P)S agar mei!m= Uner
t%e"e %ig%*o"molarit/ $onition"; t%e pgp*3 pgp*8 m!tant +orm" ie at a rate
$ompara&le +it% +il*t/pe N> +orm" +%en e:po"e to +il*t/pe PA32= In t%e $a"e
o( t%e negati'e $ontrol; a" e:pe$te; neit%er t%e +il*t/pe nor t%e pgp*3 pgp*8
+orm" +ere "!"$epti&le to t%e p/o$/anin*e5$ient PA32 m!tant 8EC=
#ig!re 4= C= elegan" P*gl/$oprotein m!tant pgp*3 pgp*8 "!ppre""e" t%e m!$, e(e$t
in t%e (a"t killing a""a/= T%irt/ to 27 L2*"tage +orm" +ere pla$e on la+n" o( t%e
ini$ate PA32 "train" gro+n on P)S mei!m; in$!&ate at >4TC an "$ore (or
ea6li'e +orm" a(ter 2 %= ,ata repre"ent t%e mean an "tanar e'iation o( t%ree
repli$ate"=
,o+nloa 5g!re to Po+erPoint
Com&ine; t%e o&"er'ation" t%at t%e m!$, m!tant e:%i&it" a e(e$t in t%e C=
elegan" (a"t killing a""a/; t%at t%e m!$, m!tant pro!$e" +il*t/pe le'el" o(
p/o$/anin an t%at pgp*3 pgp*8 +orm" are more "!"$epti&le to m!$, t%an a
p/o$/anin*e5$ient PA32 m!tant are $on"i"tent +it% t%e $on$l!"ion t%at M!$, i"
in'ol'e in t%e pro!$tion o( an e:tra$ell!lar to:in t%at i" not p/o$/anin=
T%e M!$, $atal/ti$ "erine i" reO!ire (or (a"t killing
T%e re$ent emon"tration t%at t%e E= $oli ,egP protein; t%e apparent ort%olog!e o(
M!$,; %a" $%aperone a$ti'it/ le !" to e:amine t%e re"pe$ti'e $ontri&!tion" o( t%e
proteol/ti$ an $%aperone a$ti'itie" to M!$, (!n$tion +it% re"pe$t to t%ree
p%enot/pe"; m!$oi/; temperat!re "en"iti'it/ an C= elegan" (a"t killing= To are""
t%i" O!e"tion; m!$,S>3BA +a" $on"tr!$te; in +%i$% t%e $oon en$oing t%e "erine
at t%e protea"e $atal/ti$ "ite Hetermine &/ alignment +it% E= $oli egPI +a"
m!tate to en$oe alanine H"ee E:perimental pro$e!re" (or etail"I= M!$,S>3BA;
$arrie on pla"mi pPY>>B*8; pro'ie no ete$ta&le $omplementation o( t%e
temperat!re "en"iti'it/ o( a m!$, m!tant in "tationar/ p%a"e &rot% $!lt!re" at 24TC
an; in (a$t; "lig%tl/ e:a$er&ate t%e temperat!re "en"iti'it/; +%erea" t%e +il*t/pe
m!$, $lone re"tore temperat!re re"i"tan$e (!ll/ Hata not "%o+nI= On t%e ot%er
%an; a" "%o+n in #ig= D; t%e m!$,S>3BA $lone i ela/ an partiall/ "!ppre"" t%e
on"et o( t%e m!$oi p%enot/pe in t%e m!$, "train= T%!"; alt%o!g% t%e in 'i'o
"ta&ilit/ o( t%e altere M!$, protein %a" not &een te"te; t%e eQe$t o( t%e
m!$,S>3BA $lone on m!$oi/ ini$ate" t%at M!$,S>3BA i" e:pre""e in 'i'o=
Moreo'er; t%e $orre"poning amino a$i $%ange in E= $oli ,egP i not aQe$t t%e
amo!nt o( M!$, protein or make an/ ete$ta&le $%ange in "e$onar/ "tr!$t!re
HSkorko*)lonek et al=; 3FF4L Spie"" et al=; 3FFFI= In an/ $a"e; t%e partial "!ppre""ion
o( m!$oi/ &/ m!$,S>3BA i" $on"i"tent +it% t%e %/pot%e"i" t%at M!$, %a"
$%aperone a$ti'it/ t%at $ontri&!te" to it" (!n$tion; "imilar to t%e E= $oli ,egP
protein= #inall/; m!$,S>3BA i not re"tore C= elegan" (a"t killing a$ti'it/ to a m!$,
m!tant; +%erea" +il*t/pe m!$, HpPY>>3I $omplemente (!ll/ Hata not "%o+nI=
O'erall; t%e"e re"!lt" "!gge"t t%at t%e M!$, $atal/ti$ "erine i" reO!ire (or t%e
pro!$tion o( an e:tra$ell!lar 'ir!len$e (a$torH"I ne$e""ar/ (or C= elegan" (a"t
killing=
#ig!re D= T%e PA32 m!$,S>3BA $lone HpPY>>B*8I ela/" an partiall/ $omplement"
t%e PA32 m!$, m!$oi p%enot/pe= Strain" +ere "treake onto NB M $ar&eni$illin
agar plate" an in$!&ate at 8BTC (or t%e ini$ate time"= 3; m!$, Hno gro+t% on
$ar&eni$illinIL >; m!$, pUCP3CL 8; m!$, pPY>>3L 2; m!$, pPY>>B*8=
,o+nloa 5g!re to Po+erPoint
,i"$!""ion
Top o( page
A&"tra$t
Intro!$tion
Re"!lt"
,i"$!""ion
E:perimental pro$e!re"
A$kno+legement"
Re(eren$e"
We %a'e $%ara$teri9e a 'ariet/ o( p%enot/pe" o( a P= aer!gino"a "train PA32 m!$,
m!tant= T%e prei$te PA32 M!$, protein i" 8BV ienti$al to t%e E= $oli ,egP
protein H&/ LipmanGPear"on protein alignmentI; an M!$, an ,egP appear to &e
ort%olog!e"= T%e PA32 m!$, m!tant (orm" m!$oi $olonie" an e:%i&it" in$rea"e
"en"iti'it/ to temperat!re an o:iati'e "tre""= T%e "imple"t e:planation (or t%e
m!$oi p%enot/pe i" t%at t%e lo"" o( M!$, (!n$tion lea" to t%e a$$!m!lation o(
improperl/ (ole or amage protein" Hor ot%er "tre"" "ignal"I in t%e peripla"m;
+%i$% in t!rn lea" inire$tl/ to alginate o'erpro!$tion=
In aition to m!$oi/; t%e PA32 m!$, m!tant e:%i&it" re!$e 'ir!len$e in t%ree
ramati$all/ iQerent pat%ogenG%o"t moel "/"tem"; Ara&iop"i"; C= elegan" an
mi$e= A re!$tion in 'ir!len$e o( t%e m!$, m!tant +a" al"o "een in a P= aer!gino"aG
+a: mot% H)= mellonellaI pat%ogenG%o"t moel; in +%i$% +il*t/pe PA32 /iele an
L,47 o( 3; +%erea" t%e PA32 m!$, m!tant e:%i&ite an L,47 o( C HJaner et al=;
>777I= Intere"tingl/; t%e m!$, m!tant appear" to &e impaire in t%e pro!$tion or
"e$retion o( an e:tra$ell!lar lo+*mole$!lar*+eig%t 'ir!len$e (a$tor reO!ire (or
killing C= elegan"= M!$, la$king t%e protea"e $atal/ti$ "erine HM!$,S>3BAI (aile to
$omplement t%e m!$, m!tation in t%e C= elegan" (a"t killing a""a/; "!gge"ting t%at
t%e pro!$tion o( t%e (a"t killing to:inH"I ma/ epen on t%e protea"e a$ti'it/ an
not t%e %/pot%e"i9e $%aperone a$ti'it/ o( M!$,= Ho+e'er; M!$,S>3BA i
partiall/ "!ppre"" t%e m!$oi p%enot/pe o( t%e m!$, m!tant=
T%e m!$oi p%enot/pe o( t%e m!$, m!tant le !" to e:plore t%e rele'an$e o(
alginate pro!$tion on t%e pat%ogeni$it/*relate p%enot/pe" o( t%e m!$, m!tant=
Con"eO!entl/; +e $on"tr!$te a eletion o( a ke/ alginate &io"/nt%eti$ gene; alg,;
in &ot% +il*t/pe an m!$, &a$kgro!n"= A" e:pe$te; t%e alg, eletion in t%e
m!$, &a$kgro!n a&oli"%e t%e m!$oi p%enot/pe on agar plate"= T%e a&ilit/ to
"/nt%e"i9e alginate %a no eQe$t on t%e temperat!re "en"iti'it/ or "en"iti'it/ to
H>O>= Wit% re"pe$t to pat%ogeni$it/; t%e alg, m!tation alone %a minimal or no
impa$t on 'ir!len$e in an Ara&iop"i" lea( in5ltration a""a/; t+o iQerent C= elegan"
killing a""a/" an a mo!"e t%ermal &!rn inE!r/ moel= In $ontra"t to t%e"e re"!lt";
%o+e'er; t%e alg, m!tant e:%i&ite re!$e 'ir!len$e in t%e +a: mot%
pat%ogene"i" moel H+il*t/pe PA32 /iele an L,47 o( 3; +%erea" PA32 alg,
m!tant" e:%i&ite an L,47 o( 37L Janer et al=; >777I= T%e a&"en$e o( a maEor role
(or Alg, in Ara&iop"i" an C= elegan" +a" al"o reSe$te in t%e re"!lt" o&taine
+it% t%e m!$,alg, o!&le m!tant"; +%i$% e:%i&ite "imilar le'el" o( 'ir!len$e to
t%e m!$, m!tant in t%e"e t+o moel"= Intere"tingl/; %o+e'er; t%e m!$,alg,
m!tant +a" ramati$all/ re!$e in 'ir!len$e in a mo!"e t%ermal inE!r/ moel
$ompare +it% +il t/pe or eit%er m!tant alone= In t%e +a: mot% moel; t%e
m!$,alg, o!&le m!tant al"o "%o+e an aiti'e negati'e impa$t on 'ir!len$e;
e:%i&iting an L,47 o( C7 HJaner et al=; >777I=
In "!mmar/; t%e t+o mo"t "igni5$ant 5ning" o( t%i" +ork +ere t%e o&"er'e
"/nergi"ti$ eQe$t o( t%e m!$, an alg, m!tation" on PA32 'ir!len$e in mi$e an
t%e emon"tration t%at t%e $atal/ti$ "erine o( M!$, i" e""ential (or t%e pro!$tion o(
an e:tra$ell!lar 'ir!len$e (a$torH"I reO!ire (or C= elegan" (a"t killing=
Are E= $oli egP an P= aer!gino"a m!$, ort%olog!e"-
Man/ )ram*negati'e &a$teria e:amine to ate $ontain m!ltiple egP %omolog!e";
an m!tation" in t%e"e %omolog!e" o(ten /iel 'arie p%enot/pe" HPallen an Wren;
3FFBI= P= aer!gino"a PAO3 $ontain" t+o egP %omolog!e"; m!$, an algW;
+%erea" E= $oli $ontain" egS an egP= A "ear$% o( t%e "eO!en$e PAO3 genome
H%ttpA66+++=p"e!omona"=$om6ine:=%tmlI HSto'er et al=; >777I re'eale no
aitional %ig%l/ %omologo!" (amil/ mem&er"= Se'eral o&"er'ation" "!gge"t t%at
m!$, an egP are ort%olog!e"= #ir"t; &ot% PA32 m!$, an E= $oli egP m!tant"
are temperat!re "en"iti'e HLipin"ka et al=; 3FCFL Stra!$% et al=; 3FCFI= Se$onl/;
m!$, $an partiall/ $omplement t%e temperat!re "en"iti'it/ p%enot/pe o( an E= $oli
egP m!tant= T%irl/; &ot% m!$, an egP are apparentl/ tran"$riptionall/
reg!late &/ ort%ologo!" "tre""*a$ti'ate "igma (a$tor" HAlgU an RpoE
re"pe$ti'el/L Y! et al=; 3FF4I; +%erea" t%ere i" no e'ien$e t%at algW; egP or egS
HBa"" et al=; 3FFDL Bo!$%er et al=; 3FFDL Waller an Sa!er; 3FFDI i" reg!late in a
"imilar (a"%ion= In P= aer!gino"a; m!$, i" "it!ate o+n"tream o( algU in +%at
appear" to &e an algUm!$ABC, operon= Alt%o!g% t%e E= $oli egP gene i" not linke
to rpoE; t%e E= $oli rpoE gene i" in an operon +it% t%e m!$ABC %omolog!e"; r"eABC
H,e La" Pena" et al=; 3FFBL Mi""iaka" et al=; 3FFBI=
It appear" likel/ t%at t%e %trA gene" in S= t/p%im!ri!m an Br!$ella a&ort!" an t%e
%trA6g"rA gene in Yer"inia entero$oliti$a are al"o egP ort%olog!e"; &a"e on RpoE
H"igmaEI*like promoter"; reO!irement (or temperat!re re"i"tan$e an; in t%e $a"e o(
B= a&ort!"; t%e a&ilit/ o( t%e %trA gene to $omplement E= $oli egP m!tant" HJo%n"on
et al=; 3FF3L El9er et al=; 3FF2L Roop et al=; 3FF2L Tat!m et al=; 3FF2L Li et al=; 3FFDL
Yamamoto et al=; 3FFDI=
Role o( m!$, an alg, in Ara&iop"i"; mo!"e; C= elegan" an +a: mot%
pat%ogene"i"
T%e re"!lt" reporte %ere an &/ Janer et al= H>777I "%o+ t%at M!$, i" e""ential (or
(!ll 'ir!len$e in Ara&iop"i"; mi$e; C= elegan" an +a: mot%"= T%i" i" $on"i"tent +it%
pre'io!" report" o( re!$e 'ir!len$e o( a PAO3 m!$, m!tant in a mo!"e
"epti$aemia moel HY! et al=; 3FFDI= M!tation" in pre"!mpti'e egP ort%olog!e" in
S= t/p%im!ri!m H%trAI an Y= entero$oliti$a H%trA6g"rAI al"o re"!lt in atten!ate
'ir!len$e in mo!"e moel" HJo%n"on et al=; 3FF3L Ba!mler et al=; 3FF2L Li et al=;
3FFDL Yamamoto et al=; 3FFDI= In $ontra"t; PAO3 m!tant" la$king algW; +%i$% oe"
not appear to &e an ort%olog!e o( egP; "%o+e no re!$tion in 'ir!len$e $ompare
+it% +il t/pe in a ne!tropeni$ mo!"e moel HBo!$%er et al=; 3FFDI=
In $ontra"t to m!$,; +%i$% appear" to &e generall/ reO!ire (or pat%ogene"i" in a
'ariet/ o( %o"t" an in(e$tion moel"; t%e rele'an$e o( alg, to 'ir!len$e i" m!$%
more limite= Alg, oe" not appear to &e a "igni5$ant PA32 'ir!len$e (a$tor (or
Ara&iop"i" or C= elegan" pat%ogene"i"= Moreo'er; alt%o!g% alginate i" impli$ate
a" a 'ir!len$e (a$tor in P= aer!gino"a in(e$tion" in t%e l!ng" o( $/"ti$ 5&ro"i"
patient" an mo!"e l!ng in(e$tion $learan$e moel" H)o'an an ,ereti$; 3FFDL Y!
et al=; 3FFCI; o!r re"!lt" ini$ate t%at alginate i" not an important 'ir!len$e (a$tor in
a mo!"e t%ermal inE!r/ moel= T%i" i" $on"i"tent +it% a pre'io!" report t%at a P=
aer!gino"a PAO3 alg, m!tant +a" not impaire in 'ir!len$e in a &!rne mo!"e
moel H)ol&erg et al=; 3FF4I= T%e"e re"!lt" "!gge"t t%at; in mammalian
pat%ogene"i"; alginate ma/ onl/ &e an important 'ir!len$e (a$tor in $%roni$
in(e$tion" t%at in'ol'e t%e (ormation o( &io5lm" "!$% a" in t%e l!ng" o( $/"ti$ 5&ro"i"
patient"= Alt%o!g% alginate +a" not reO!ire (or pat%ogene"i" in t%e plant;
nematoe an mo!"e moel"; alginate oe" appear to &e important (or a$!te
in(e$tion in +a: mot%"; in +%i$% t%e alg, m!tant e:%i&ite a 37*(ol re!$tion in
'ir!len$e; "%o+ing an L,47 o( 37 'er"!" an L,47 o( 3 (or +il*t/pe PA32 HJaner
et al=; >777I=
In $ontra"t to o!r 5ning t%at alginate oe" not appear to &e important (or PA32
pat%ogene"i" in Ara&iop"i"; Y! et al= H3FFFI o&"er'e a "igni5$ant rop in 'ir!len$e
in an alginate m!tant o( t%e plant pat%ogen P= "/ringae p'= "/ringae 84>4 in it"
nat!ral %o"t &ean= In aition; ot%er e:opol/"a$$%arie" %a'e al"o &een reporte to
pla/ important role" in t%e 'ir!len$e o( a 'ariet/ o( plant pat%ogen" HLeig% an
Coplin; 3FF>L Ne+man et al=; 3FF2L Conemine et al=; 3FFFI=
,o Alg, an M!$, pro'ie prote$tion (rom o:iati'e "tre""-
T%e o&"er'ation t%at PA32 an PAO3 m!$, m!tant" e:%i&it in$rea"e "en"iti'it/ to
o:iati'e "tre"" in 'itro HBo!$%er et al=; 3FFDI i" $on"i"tent +it% t%e %/pot%e"i9e
role o( ,egP ort%olog!e" in mammalian pat%ogen" a" pro'iing prote$tion (rom t%e
eleterio!" eQe$t" o( t%e ma$rop%age o:iati'e &!r"t HJo%n"on et al=; 3FF3I=
Ho+e'er; in plant"; re$ent e'ien$e "!gge"t" t%at H>O> pro!$e in t%e plant
e(en$e*a""o$iate o:iati'e &!r"t in to&a$$o lea'e" an potato t!&er" i" not
ire$tl/ eleterio!" to in'aing mi$ro&e"; in$l!ing an Er+inia $%r/"ant%emi o:/R
m!tant t%at i" %/per"!"$epti&le to H>O> HMig!el et al=; >777I= I( t%i" re"!lt pro'e" to
&e generall/ tr!e in plantGpat%ogen intera$tion"; t%e role o( M!$, in plant
pat%ogene"i" +o!l (!n$tion &/ "ome me$%ani"m ot%er t%an prote$ting again"t an
o:iati'e &!r"t=
Alginate %a" al"o &een reporte to pro'ie prote$tion again"t o:iati'e "tre"";
in$l!ing "$a'enging %/po$%lorite an rea$ti'e o:/gen "pe$ie" generate &/
ma$rop%age" or ne!trop%il" HLearn et al=; 3FCBL Simp"on et al=; 3FCFI= Alt%o!g% o!r
in 'itro a""a/ "!gge"t" t%at alginate oe" not prote$t (rom H>O> "tre""; it remain"
po""i&le t%at it $o!l pro'ie prote$tion in 'i'o; a" t%e timing an le'el" o( alginate
e:pre""ion an t%e t/pe" o( rea$ti'e o:/gen "pe$ie" en$o!ntere +o!l pro&a&l/ &e
iQerent= In t%e &!rne mo!"e in(e$tion moel; alginate apparentl/ pla/" a limite
Hi( an/I role in 'ir!len$e in +il*t/pe P= aer!gino"a; &!t t%e (a$t t%at t%e m!$,alg,
o!&le m!tant +a" ramati$all/ le"" 'ir!lent t%at t%e m!$, "ingle m!tant "!gge"t"
t%at t%e o'erpro!$tion o( alginate in t%e m!$, m!tant ma/ partiall/ $ompen"ate
(or t%e lo"" o( M!$, (!n$tion= One +a/ in +%i$% alginate o'erpro!$tion $o!l
$ompen"ate (or M!$, (!n$tion i" &/ prote$ting peripla"mi$ protein" (rom o:iati'e
amage; t%ere&/ re!$ing t%e nee (or t%e repair an "al'age (!n$tion" o( M!$,= A
"imilar me$%ani"m $o!l &e operating in t%e +a: mot% moel; gi'en t%e (a$t t%at
t%e m!$,alg, m!tant al"o e:%i&it" ramati$all/ lo+er 'ir!len$e t%an eit%er t%e
m!$, or t%e alg, m!tant" alone=
T%e M!$, $atal/ti$ "erine i" reO!ire (or t%e "/nt%e"i" or e:port o( a lo+*mole$!lar*
+eig%t to:in
T%ere are at lea"t t+o $la""e" o( to:in" reO!ire (or (a"t killing o( C= elegan"= One
$la"" in$l!e" p%ena9ine"; +%erea" t%e ientit/ o( t%e "e$on $la"" i" !nkno+n= T%e
PA32 m!$, m!tant i" impaire in (a"t killing; /et pro!$e" t%e p%ena9ine p/o$/anin
at +il*t/pe or %ig%er le'el"= In aition; pgp*3 pgp*8 m!tant +orm" e:%i&it
en%an$e "en"iti'it/ to t%e m!$, "train= T%e"e ata ini$ate t%at t%e m!$, m!tant
&elong" to t%e $la"" o( PA32 m!tant" impaire in C= elegan" (a"t killing t%at are
e(e$ti'e in a to:in i"tin$t (rom p/o$/anin= #!rt%ermore; +e %a'e pro'ie
e'ien$e t%at t%e pro!$tion o( t%i" to:in ma/ &e epenent on t%e proteol/ti$
a$ti'it/ o( M!$,; +it% t%e $a'eat t%at t%e "ta&ilit/ o( t%e pre"!mpti'e proteol/ti$all/
ina$ti'e m!tant M!$, protein %a" not &een etermine= #or e:ample; M!$, $o!l
&e in'ol'e in t%e generation o( lo+*mole$!lar*+eig%t to:i$ peptie" $lea'e (rom a
larger pre$!r"or protein; "imilar to t%e (!n$tion o( a egP %omolog!e in La$to$o$$!"
la$ti" t%at i" reO!ire (or t%e proteol/ti$ a$ti'ation o( an e:porte a!tol/"in HPoO!et
et al=; >777I= Alternati'el/; t%e lo"" o( M!$, proteol/ti$ a$ti'it/ ma/ re"!lt in
in$rea"e $ell en'elope "tre""; +%i$% $o!l %a'e pleiotropi$ eQe$t" on t%e
e:pre""ion o( a 'ariet/ o( gene"; in$l!ing "ome e""ential (or (a"t killing= We are
$!rrentl/ p!r"!ing e:periment" to ienti(/ t%e to:inH"I t%at are e5$ient in PA32
m!$, m!tant"= It +ill &e o( intere"t to etermine +%et%er t%e"e p!tati'e to:in" pla/
a "igni5$ant role in 'ir!len$e in ot%er %o"t"=
E:perimental pro$e!re"
Top o( page
A&"tra$t
Intro!$tion
Re"!lt"
,i"$!""ion
E:perimental pro$e!re"
A$kno+legement"
Re(eren$e"
Strain"; pla"mi" an gro+t% $onition"
P"e!omona" aer!gino"a "train UCBPP*PA32 Hre(erre to a" PA32 (or "impli$it/I i" a
%!man $lini$al i"olate $apa&le o( $a!"ing i"ea"e in animal" an plant" HRa%me
et al=; 3FF4L Ma%aEan*Miklo" et al=; 3FFFL Tan et al=; 3FFFaI= PA32 8EC $ontain" a
Tnp%oA in"ertion m!tation in t%e p%ena9ine &io"/nt%eti$ gene p%9B HMa%aEan*
Miklo" et al=; 3FFFLI= All $loning "tep" +ere $arrie o!t in E= $oli ,H4 HdpirI HNolter
et al=; 3FBCI= Wil*t/pe E= $oli "train NS>B> an t%e i"ogeni$ m!tant egP23 H"train
NS2B2I %a'e &een e"$ri&e pre'io!"l/ HStra!$% et al=; 3FCFI= T%e pla"mi" !"e
an $on"tr!$te in t%e $o!r"e o( t%i" +ork are li"te in Ta&le >= Ba$terial "train"
+ere gro+n at 8BTC in L!ria &rot% HLBL Miller; 3FB>I; NingJ" B or NingJ" A meia HNB;
NAL Ning et al=; 3F42I; MF minimal meia HMiller; 3FB>I or Ba$to P"e!omona"
i"olation agar HPIAL ,i($oI a" note= Anti&ioti$ $on$entration" !"e (or PA32 +ereA
ri(ampi$in; 377 Wg mlX3L gentami$in; 87 Wg mlX3L $ar&eni$illin; 877 Wg mlX3L
kanam/$in; >77 Wg mlX3=
Ta&le >= Strain" an pla"mi" !"e in t%i" "t!/=
Strain or
pla"mi
,e"$ription So!r$e or re(eren$e
pUC3C63F Cloning 'e$tor"; ampr
Yani"$%*Perron et al=
H3FC4I
pUCP3C63F
Cloning 'e$tor" t%at repli$ate "ta&l/ in
P"e!omona"; ampr
S$%+ei9er H3FF3I
pC1,22>
Po"iti'e "ele$tion "!i$ie 'e$tor (or marker
e:$%ange; ampr
,onnen&erg an
Naper H3FF3I
pUCB)
So!r$e o( aa$C3 gentami$in $a""ette Haa$C3 +a"
$lone (rom pPC337 into pUCBI
S= Lor/
pH3>D Co"mi $lone $arr/ing PA32 m!$, T%i" +ork
pPY>73 PA32 algUm!$ABC, in pUC3F T%i" +ork
pPY>72 m!$C an 4 en o( m!$, in pUC3F T%i" +ork
pPY>74 m!$,AAaa$C3 in pUC3F T%i" +ork
pPY>7D m!$,AAaa$C3 in pC1,22> T%i" +ork
pAL)e2PUC Internal in (rame eletion o( alg, in pUC3F T%i" +ork
pAL)e2C1, Internal in (rame eletion o( alg, in pC1,22> T%i" +ork
pPY>>7 PA32 m!$, in pUC3F T%i" +ork
pPY>>3 PA32 m!$, in pUCP3C T%i" +ork
Ta&le >= Strain" an pla"mi" !"e in t%i" "t!/=
Strain or
pla"mi
,e"$ription So!r$e or re(eren$e
pPY>>> PA32 algUm!$ABC, in pBR8>> T%i" +ork
pPY>>D PA32 alg, in pUCP3F T%i" +ork
pPY>>B*8 PA32 m!$, Ser*>3BAla in pUCP3C T%i" +ork
Cloning an ,NA "eO!en$e anal/"i" o( a P= aer!gino"a PA32 egP %omolog!e
A egP ,NA pro&e +a" generate &/ IPCR (rom a egPAATnp%oA m!tant "train
HLS2DI o( P= "/ringae p'= ma$!li$ola ES28>D HSte'en"; 3FFCI= C%romo"omal ,NA
(rom t%i" "train +a" ige"te +it% Hin$II; t%e re"!lting (ragment" $ir$!lari9e &/
ligation; t%en !"e a" template" (or IPCR +it% primer" "pe$i5$ to t%e 4 en o(
Tnp%oA= T%e Tnp%oA primer" !"e +ereA 4*AATATC)CCCT)A)CA*8 HP3IL 4*
ACTCACTAT)C)CT)AATAA*8 HP>I= A 234 &p PCR pro!$t +a" o&taine an
"eO!en$e= It in$l!e >24 &p o( egP Sanke on eit%er "ie &/ F7 &p an C7 &p o(
Tnp%oA "eO!en$e= T%i" PCR pro!$t +a" la&elle an !"e to pro&e a $o"mi li&rar/
o( PA32 $%romo"omal ,NA HRa%me et al=; 3FF4I= One $o"mi $lone; pH3>D; t%at
%/&rii9e to t%e pro&e +a" anal/"e (!rt%er an (o!n to $ontain a 4=8 k&p SalI
(ragment t%at %/&rii9e to t%e P= "/ringae egP pro&e= T%e 4=8 k&p SalI (ragment
+a" "!&$lone into pUC3F to /iel pPY>73= A 3=8 k&p R%oIGSalI "!&$lone o( t%e SalI
(ragment t%at al"o %/&rii9e to t%e P= "/ringae egP pro&e +a" "eO!en$e an
(o!n to $ontain a portion o( a gene %omologo!" to E= $oli egP an to ot%er kno+n
egP %omolog!e"= W%en t%e aEa$ent E$oRIGR%oI (ragment +a" "eO!en$e; t%e
E$oRIGR%oI an R%oIGSalI (ragment" +ere (o!n to $ontain t%e entire egP
%omolog!e OR#= To te"t +%et%er t%e PA32 egP %omolog!e la/ immeiatel/
o+n"tream o( t%e algUm!$ABC $l!"ter; t+o primer"; ,E)B an MUCB3; +ere !"e
to ampli(/ a prei$te 4C7 &p (ragment (rom pPY>73= ,E)B H4*
AC)TAC)TAC)TAC)TAC)TI $orre"pon" to t%e 4 en o( PA32 m!$,; an MUCB3 H4*
A)AT))T)ACC)TC)TC)I $orre"pon" to t%e PAO3 m!$B "eO!en$e=
Con"tr!$tion o( a PA32 m!$, m!tation &/ in"ertion +it% t%e gentami$in re"i"tan$e
$a""ette
A 3=4 k&p P"tI (ragment $ontaining t%e 4 en o( t%e PA32 egP %omolog!e pl!"
!p"tream "eO!en$e" +a" "!&$lone (rom pPY>73 into pUC3F to /iel pPY>72= A SalI
(ragment $ontaining t%e gentami$in re"i"tan$e gene aa$C3 (rom pUCB) +a"
"!&$lone into t%e R%oI "ite earl/ in t%e egP gene to /iel pPY>74= #lanking
re"tri$tion "ite"; SalI an Sp%I; in t%e pUC3F pol/linker +ere !"e to "!&$lone t%i"
$on"tr!$t (rom pPY>74 into $orre"poning "ite" in t%e po"iti'e "ele$tion "!i$ie
'e$tor pC1,22> H,onnen&erg an Naper; 3FF3I to generate pPY>7D= pPY>7D +a"
t%en !"e to repla$e t%e +il*t/pe $op/ o( egP &/ marker e:$%ange a" e"$ri&e
pre'io!"l/ H,onnen&erg an Naper; 3FF3I=
Con"tr!$tion o( m!$, $omplementing $lone"
pPY>>7
T%e >=7 k&p m!$,*$ontaining E$oRIGSalI (ragment (rom pPY>73 +a" "!&$lone into
t%e $orre"poning "ite" in pUC3F to /iel pPY>>7=
pPY>>3
T%e >=7 k&p m!$,*$ontaining E$oRIGSalI (ragment (rom pPY>73 +a" "!&$lone into
t%e $orre"poning "ite" in pUCP3C to /iel pPY>>3 +it% m!$, e:pre""ion ri'en &/
t%e la$U promoter=
pPY>>>
T%e 4=8 k&p SalI (ragment (rom pPY>73 $ontaining t%e entire algUm!$ABC, $l!"ter
+a" "!&$lone into pBR8>> !"ing t%e HinIII an BamHI Sanking "ite" in pPY>73
an t%e $orre"poning "ite" in t%e tetra$/$line re"i"tan$e gene o( pBR8>>;
generating pPY>>>=
pPY>>B*8
T%e $oon en$oing t%e $atal/ti$ "erine o( M!$, HSer*>3B; n!m&ere (rom (!ll*
lengt% or !npro$e""e M!$,I +a" m!tate to en$oe alanine in pPY>>7 !"ing t%e
Stratagene P!ikC%ange "ite*ire$te m!tagene"i" kit= T%e 7=B k&p R%oIGP"tI
(ragment $ontaining t%i" m!tation +a" "eO!en$e to en"!re t%at t%ere +ere no
aitional $%ange" an t%en !"e to repla$e t%e $orre"poning (ragment in pPY>>3=
Pla"mi" (or $omplementation +ere ele$troporate into PA32; PA32 m!$,; PA32
m!$,alg,; E= $oli HNS>B>I an E= $oli egP23 HNS2B2I HStra!$% et al=; 3FCFI=
Con"tr!$tion o( PA32 alg,e2
#lanking "eO!en$e" o( t%e alg, gene +ere ampli5e &/ PCR (rom PA32
$%romo"omal ,NA !"ing primer" $orre"poning to p!&li"%e P= aer!gino"a "train
PAO3 alg, "eO!en$e"= T%e (ollo+ing primer" +ere !"eA 4*
T)CTA)TCTA)A)TTC)TCT)CAA)TCATTC)) HAL)4IL 4*
C)))ATCCAAA)AT)CT)ATTC)C ATC) HAL)FIL 4*
AT)C)A)CTC)CA)AATTCATT))T))T)A)) HAL)38IL 4*
CC))ATCC)A)CT)TTC)TC)ACCT))T) HAL)32I= To (a$ilitate "!&"eO!ent $loning;
ea$% primer +a" e"igne to $ontain a re"tri$tion "ite at it" 4 enA AL)4; R&aIL
AL)F an AL)32; BamHIL an AL)38; Sa$I= AL)4GAL)F H4 en o( alg,I an AL)32G
AL)38 H8 en o( alg,I primer pair" +ere !"e to ampli(/ B47 an F77 &p
(ragment"; re"pe$ti'el/; Sanking t%e alg, gene= T%e"e PCR*ampli5e (ragment"
+ere $lone into t%e R&aI an Sa$I "ite" in pUC3F a" (ollo+" to generate
pAL)e2PUCA R&aIGAL)4GAL)FGBamHI an BamHIGAL)32GAL)38GSa$I= T%i"
$on"tr!$t $ontain" a 3=C k&p $ontig!o!" $lone in +%i$% all &!t t%e 5r"t "i: an la"t
8D $oon" o( alg, are elete= T%e BamHI "ite linking t%e !p"tream an
o+n"tream (ragment" a" t+o aitional $oon" an maintain" t%e alg, reaing
(rame= #inall/; t%e alg,e2 $on"tr!$t +a" "!&$lone into t%e R&aI an Sa$I "ite" in
pC1,22> to generate pAL)e2C1,; +%i$% +a" t%en !"e to repla$e t%e +il*t/pe
$op/ o( alg, in PA32 an PA32 m!$, a" e"$ri&e pre'io!"l/ H,onnen&erg an
Naper; 3FF3I= Bot% $on"tr!$t" +ere $on5rme &/ PCR ampli5$ation o( $%romo"omal
,NA=
Con"tr!$tion o( an alg, $omplementing $lone
T%e alg, gene +a" ampli5e HE:pan Hig% #ielit/ PCR "/"temL Boe%ringer
Mann%eimI (rom PA32 $%romo"omal ,NA !"ing t%e AL)4 an AL)38 primer" H"ee
a&o'eI= T%e ampli5e pro!$t +a" $lone into t%e pUCP3F R&aIGSa$I "ite" "!$% t%at
e:pre""ion o( t%e alg, gene +a" ri'en &/ t%e pla"mi la$U promoter=
Temperat!re "en"iti'it/ gro+t% a""a/"
O'ernig%t $!lt!re" H4 mlI +ere gro+n in NB mei!m at 8BTC= AliO!ot" H3 mlI o(
t%e"e $!lt!re" +ere t%en in$!&ate on a t!&e roller at 24TC= Initial $(!" an $(!"
a(ter 2 % +ere etermine &/ "erial il!tion an plating on NB "!pplemente +it%
t%e appropriate anti&ioti$"=
H/rogen pero:ie "en"iti'it/ a""a/"
Ea$% "train +a" gro+n o'ernig%t in LB pl!" ri(ampi$in at 8BTC= T%e (ollo+ing
morning; 477 Wl o( ea$% $!lt!re +a" "!&$!lt!re into 4 ml o( LB an gro+n (or 4GD %
to earl/ "tationar/ p%a"e= AliO!ot" H8 mlI o( ea$% "train +ere %ar'e"te &/
$entri(!gation an re"!"pene in 37 mM MgSO2 at an O,D77 o( 7=>= T%i"
"!"pen"ion +a" ae H4 Wl mlX3 3=4V lo+ melt agaro"e pre*eO!ili&rate at 8BTCI
an !"e to po!r 4 ml o'erla/" on LB plate"= T%e"e o'erla/" +ere allo+e to "olii(/
(or [> % at room temperat!re= A "terile 5lter paper i"k HS$%lei$%er an S$%!ell no=
B27*E; 362 in$%I +a" t%en pla$e in t%e mile o( ea$% plate; an 37 Wl o( 4V H>O>
+a" pipette onto t%e i"k= Plate" +ere in$!&ate o'ernig%t at 8BTC; an 9one" o(
in%i&ition +ere mea"!re on t%e (ollo+ing a/=
Plant pat%ogeni$it/ a""a/"
T%e 'ir!len$e o( PA32 "train" +a" te"te in Ara&iop"i" e$ot/pe LL gro+n in a
green%o!"e an "!&"eO!entl/ in a plant gro+t% $%am&er a" e"$ri&e pre'io!"l/
HRa%me et al=; 3FF4I= Sat!rate $!lt!re" o( PA32 +ere gro+n in LB an prepare a"
e"$ri&e pre'io!"l/ HRa%me et al=; 3FF4I= Ba$terial "!"pen"ion" at an O,D77 o(
7=77> +ere in5ltrate into ini'i!al lea'e" o( 4* to D*+eek*ol plant" a" e"$ri&e
pre'io!"l/ (or t%e Ara&iop"i" pat%ogen P= "/ringae H,ong et al=; 3FF3I= Plant" +ere
t%en in$!&ate at >FTC an F7G377V %!miit/ in a plant gro+t% $%am&er= Ba$terial
proli(eration in lea'e" +a" etermine &/ taking t+o lea( p!n$%e" per in5ltrate lea(
(ollo+e &/ grining ea$% pair o( p!n$%e" in >77 Wl o( 37 mM MgSO2 in mi$ro(!ge
t!&e" an il!ting an plating (or $(!" on appropriate "ele$ti'e meia=
Mo!"e (!ll*t%i$kne"" "kin t%ermal &!rn moel
Si:*+eek*ol male ANR6J mi$e HJa$k"on La&oratorie"I +eig%ing &et+een >4 an 87 g
+ere gi'en a 4V total "!r(a$e area &!rn on t%eir a&ominal "kin a" e"$ri&e
pre'io!"l/ HSte'en" et al=; 3FF2I= In mortalit/ "t!ie"; immeiatel/ a(ter t%e &!rn;
mi$e +ere inEe$te +it% 4 \ 374 PA32 $ell"; an t%e n!m&er o( animal" t%at
e'elope "ep"i" +a" monitore ea$% a/ (or 37 a/"= Animal "t!/ proto$ol" +ere
re'ie+e an appro'e &/ t%e S!&$ommittee on Animal St!ie" o( t%e
Ma""a$%!"ett" )eneral Ho"pital= #i"%er e:a$t te"t +it% t%e Bon(erroni $orre$tion +a"
!"e (or "tati"ti$al anal/"i" H%ttpA66%ome=$lara=net6"i"a65"%r%lp=%tmI=
Caenor%a&iti" elegan" killing a""a/"
Caenor%a&iti" elegan" killing a""a/" +ere $arrie o!t a" e"$ri&e !"ing L2*"tage
Bri"tol "train N> +il*t/pe; (er*3 HA$%an9ar an War; 3FFBI or NL387 pgp*3Hpk3BIL
pgp*8Hpk3CI HBroek" et al=; 3FF4I +orm" on P)S HpeptoneGgl!$o"eG"or&itolL (a"t
killingI or N) Hnematoe gro+t%L "lo+ killingI plate" HMa%aEan*Miklo" et al=; 3FFFL
Tan et al=; 3FFFaI= All a""a/" +ere $arrie o!t at >4TC=
P/o$/anin a""a/"
O'ernig%t $!lt!re" o( PA32 an eri'ati'e" +ere gro+n in NingJ" A M 37 WM #eCl8
+it% appropriate anti&ioti$"= O'ernig%t $!lt!re" H8 WlI +ere "!&$!lt!re into 8 ml o(
(re"% NingJ" A M 37 WM #eCl8 +it%o!t anti&ioti$" Hri(ampi$in inter(ere" +it% t%e
$olorimetri$ a""a/I an gro+n (or 3DG>2 % on a t!&e roller at 8BTC= #or ea$% $!lt!re;
3 ml +a" $entri(!ge; an p/o$/anin a""a/" +ere $on!$te on "!pernatant" a"
e"$ri&e pre'io!"l/ HE""ar et al=; 3FF7I= #or ea$% "train te"te; O!ar!pli$ate
$!lt!re" +ere gro+n an a""a/e= P/o$/anin pro!$tion +a" normali9e to
$(! mlX3; t%e mean Han S,I $al$!late an repre"ente a" a per$entage o( +il*
t/pe pro!$tion=
A$kno+legement"
Top o( page
A&"tra$t
Intro!$tion
Re"!lt"
,i"$!""ion
E:perimental pro$e!re"
A$kno+legement"
Re(eren$e"
We t%ank S%alina Ma%aEan*Miklo" (or %elp(!l i"$!""ion"; an )eral Pier an J!lie
Stone (or $riti$al reaing o( t%e man!"$ript= P=Y= +a" "!pporte &/ an Ameri$an
Can$er So$iet/ #ello+"%ip an NIH Training )rant T8> )M7B784= T%i" +ork +a"
"!pporte in part &/ a grant (rom A'enti" SA to Ma""a$%!"ett" )eneral Ho"pital=
Re(eren$e"
Top o( page
A&"tra$t
Intro!$tion
Re"!lt"
,i"$!""ion
E:perimental pro$e!re"
A$kno+legement"
Re(eren$e"
3
A$%an9ar; W=E= 0 War; S= H3FFBI A nematoe gene reO!ire (or "perm 'e"i$le
(!"ion= J Cell S$i 337A 37B8G37C3
P!&Me;
C%emPort;
We& o( S$ien$ef
>
Al'are9; M=E=; Pennell; R=I=; MeiEer; P=*J=; I"%ika+a; A=; ,i:on; R=A=; Lam&; C= H3FFCI
Rea$ti'e o:/gen intermeiate" meiate a "/"temi$ "ignal net+ork in t%e
e"ta&li"%ment o( plant imm!nit/= Cell F>A BB8GBC2
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef
8
Ba""; S=; )!; P=; C%ri"ten; A= H3FFDI M!lti$op/ "!ppre""or" o( pr$ m!tant E"$%eri$%ia
$oli in$l!e t+o HtrA H,egPI protea"e %omolog" HH%oABI; ,k"A; an a tr!n$ate
R3pA= J Ba$teriol 3BCA 3342G33D3
P!&Me;
C%emPort;
We& o( S$ien$ef
2
Ba!mler; A=J=; N!"ter"; J=)=; StoEilEko'i$; I=; HeQron; #= H3FF2I Salmonella t/p%im!ri!m
lo$i in'ol'e in "!r'i'al +it%in ma$rop%age"= In(e$t Imm!n D>A 3D>8G3D87
P!&Me;
C%emPort;
We& o( S$ien$ef
4
Bo!$%er; J=C=; Martine9*Sala9ar; J=; S$%!rr; M=J=; M!; M=H=; Y!; H=; ,ereti$; 1= H3FFDI
T+o i"tin$t lo$i aQe$ting $on'er"ion to m!$oi/ in P"e!omona" aer!gino"a in
$/"ti$ 5&ro"i" en$oe %omolog" o( t%e "erine protea"e HtrA= J Ba$teriol 3BCA 433G
4>8
P!&Me;
C%emPort;
We& o( S$ien$ef
D
Broek"; A=; Jan""en; H=W=R=M=; Cala(at; J=; Pla"terk; R=H=A= H3FF4I A P*gl/$oprotein
prote$t" Caenor%a&iti" elegan" again"t nat!ral to:in"= EMBO J 32A 3C4CG3CDD
P!&Me;
C%emPort;
We& o( S$ien$ef
B
Conemine; )=; Ca"tillo; A=; Pa""eri; #=; Enar; C= H3FFFI T%e Pe$T repre""or
$oreg!late" "/nt%e"i" o( e:opol/"a$$%arie" an 'ir!len$e (a$tor" in Er+inia
$%r/"ant%emi= Mol PlantGMi$ro&e Intera$t 3>A 24G4>
Cro""Re(;
P!&Me;
We& o( S$ien$ef
C
Connoll/; L=; ,e La" Pena"; A=; Al&a; B=M=; )ro""; C=A= H3FFBI T%e re"pon"e to
e:tra$/topla"mi$ "tre"" in E"$%eri$%ia $oli i" $ontrolle &/ partiall/ o'erlapping
pat%+a/"= )ene" ,e' 33A >73>G>7>3
Cro""Re(;
P!&Me;
We& o( S$ien$ef
F
,e La" Pena"; A=; Connoll/; L=; )ro""; C=A= H3FFBI T%e "igmaE*meiate re"pon"e to
e:tra$/topla"mi$ "tre"" in E"$%eri$%ia $oli i" tran"!$e &/ R"eA an R"eB; t+o
negati'e reg!lator" o( "igmaE= Mol Mi$ro&iol >2A 8B8G8C4
,ire$t LinkA
A&"tra$t
P,#H4BFNI
37
,ereti$; 1=; S$%!rr; M=J=; Bo!$%er; J=C=; Martin; ,=W= H3FF2I Con'er"ion o(
P"e!omona" aer!gino"a to m!$oi/ in $/"ti$ 5&ro"i"A en'ironmental "tre"" an
reg!lation o( &a$terial 'ir!len$e &/ alternati'e "igma (a$tor"= J Ba$teriol 3BDA >BB8G
>BC7
P!&Me;
C%emPort;
We& o( S$ien$ef
33
,e1rie"; C=A= 0 O%man; ,=E= H3FF2I M!$oi*to*nonm!$oi $on'er"ion in alginate*
pro!$ing P"e!omona" aer!gino"a o(ten re"!lt" (rom "pontaneo!" m!tation" in
algT; en$oing a p!tati'e alternate "igma (a$tor; an "%o+" e'ien$e (or
a!toreg!lation= J Ba$teriol 3BDA DDBBGDDCB
P!&Me;
C%emPort;
We& o( S$ien$ef
3>
,ong; R=; Minrino"; M=; ,a'i"; N=R=; A!"!&el; #=M= H3FF3I In!$tion o( Ara&iop"i"
t%aliana e(en"e gene" &/ 'ir!lent an a'ir!lent P"e!omona" "/ringae "train" an
&/ a $lone a'ir!len$e gene= Plant Cell 8A D3GB>
Cro""Re(;
P!&Me;
We& o( S$ien$ef
38
,onnen&erg; M=S= 0 Naper; J=B= H3FF3I Con"tr!$tion o( an eae eletion m!tant o(
enteropat%ogeni$ E"$%eri$%ia $oli &/ !"ing a po"iti'e*"ele$tion "!i$ie 'e$tor= In(e$t
Imm!n 4FA 2837G283B
P!&Me;
C%emPort;
We& o( S$ien$ef
32
El9er; P=H=; P%illip"; R=W=; No'a$%; M=E=; Petter"on; N=M=; Roop; R=M= H3FF2I
C%ara$teri9ation an geneti$ $omplementation o( a Br!$ella a&ort!" %ig%*
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P!&Me;
C%emPort;
We& o( S$ien$ef
34
El9er; P=H=; P%illip"; R=W=; Ro&ert"on; )=T=; Roop; R=M=; II H3FFDI T%e HtrA "tre""
re"pon"e protea"e $ontri&!te" to re"i"tan$e o( Br!$ella a&ort!" to killing &/ m!rine
p%ago$/te"= In(e$t Imm!n D2A 2C8CG2C23
P!&Me;
C%emPort;
We& o( S$ien$ef
3D
E""ar; ,=W=; E&erl/; L=; Haero; A=; Cra+(or; I=P= H3FF7I Ienti5$ation an
$%ara$teri9ation o( gene" (or a "e$on ant%ranilate "/nt%a"e in P"e!omona"
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impli$ation"= J Ba$teriol 3B>A CC2GF77
P!&Me;
C%emPort;
We& o( S$ien$ef
3B
)ol&erg; J=B=; )orman; W=L=; #l/nn; J=L=; O%man; ,=E= H3FF8I A m!tation in algN
permit" tran" a$ti'ation o( alginate pro!$tion &/ algT in P"e!omona" "pe$ie"= J
Ba$teriol 3B4A 3878G387C
P!&Me;
C%emPort;
We& o( S$ien$ef
3C
)ol&erg; J=B=; Co/ne; M=J=; Neel/; A=N=; Holer; I=A= H3FF4I A'ir!len$e o( a
P"e!omona" aer!gino"a algC m!tant in a &!rne*mo!"e moel o( in(e$tion= In(e$t
Imm!n D8A 23DDG23DF
P!&Me;
C%emPort;
We& o( S$ien$ef
3F
)o'an; J=R= 0 ,ereti$; 1= H3FFDI Mi$ro&ial pat%ogene"i" in $/"ti$ 5&ro"i"A m!$oi
P"e!omona" aer!gino"a an B!rk%oleria $epa$ia= Mi$ro&iol Re' D7A 48FG4B2
P!&Me;
C%emPort;
We& o( S$ien$ef Time" CiteA BC8
>7
Her"%&erger; C=,=; Ye; R=W=; Par"ek; M=R=; Rie; U=,=; C%akra&art/; A=M= H3FF4I T%e
algT HalgUI gene o( P"e!omona" aer!gino"a; a ke/ reg!lator in'ol'e in alginate
&io"/nt%e"i"; en$oe" an alternati'e "igma (a$tor H"igma EI= Pro$ Natl A$a S$i USA
F>A BF23GBF24
Cro""Re(;
P!&Me;
We& o( S$ien$ef Time" CiteA F7
>3
Higgin"; C=#= H3FF>I ABC tran"porter"; (rom mi$roorgani"m" to man= Ann! Re' Cell
Biol CA DBG338
Cro""Re(;
P!&Me;
We& o( S$ien$ef Time" CiteA >284
>>
Higgin"; C=#= H3FF4I T%e ABC o( $%annel reg!lation= Cell C>A DF8GDFD
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef
>8
It9%aki; H=; Na'e%; L=; Lina%l; M=; Cook; M=; Aam; U= H3FFCI Ienti5$ation an
$%ara$teri9ation o( ,egP; a "erine protea"e a""o$iate +it% t%e l!minal "ie o( t%e
t%/lakoi mem&rane= J Biol C%em >B8A B7F2GB7FC
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef
>2
Janer; )=; Ra%me; L=)=; A!"!&el; #=M= H>777I Po"iti'e $orrelation &et+een 'ir!len$e
o( P"e!omona" aer!gino"a m!tant" in mi$e an in"e$t"= J Ba$teriol 3C>A 8C28G
8C24
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef
>4
Jo%n"on; N=; C%arle"; I=; ,o!gan; )=; Pi$kar; ,=; OJ)aora; P=; )eorgopo!lo"; C= ; et al=
H3FF3I T%e role o( a "tre""*re"pon"e protein in Salmonella t/p%im!ri!m 'ir!len$e=
Mol Mi$ro&iol 4A 273G27B
,ire$t LinkA
A&"tra$t
P,#H8>B7NI
Re(eren$e"
>D
Ning; E=O=; War; M=N=; Rane/; ,=E= H3F42I T+o "imple meia (or t%e emon"tration
o( p%/$o$/nin an S!ore"$ein= J La& Clin Me 22A 873G87B
P!&Me;
We& o( S$ien$ef
>B
Nolter; R=; In!9!ka; M=; Helin"ki; ,=R= H3FBCI Tran"$omplementation*epenent
repli$ation o( a lo+ mole$!lar +eig%t origin (ragment (rom pla"mi RDN= Cell 34A
33FFG3>7C
Cro""Re(;
P!&Me;
We& o( S$ien$ef
>C
Learn; ,=B=; Bre"tel; E=P=; Seet%arama; S= H3FCBI H/po$%lorite "$a'enging &/
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P!&Me;
We& o( S$ien$ef
>F
Leig%; J=A= 0 Coplin; ,=L= H3FF>I E:opol/"a$$%arie" in plantG&a$terial intera$tion"=
Ann! Re' Mi$ro&iol 2DA 87BG82D
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef
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Li; S=R=; ,orrell; N=; E'ere"t; P=H=; ,o!gan; )=; Wren; B=W= H3FFDI Con"tr!$tion an
$%ara$teri9ation o( a Yer"inia entero$oliti$a OAC %ig%*temperat!re reO!irement H%trAI
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P!&Me;
C%emPort;
We& o( S$ien$ef
83
Lipin"ka; B=; #a/et; O=; Bair; L=; )eorgopo!lo"; C= H3FCFI Ienti5$ation;
$%ara$teri9ation; an mapping o( t%e E"$%eri$%ia $oli %trA gene; +%o"e pro!$t i"
e""ential (or &a$terial gro+t% onl/ at ele'ate temperat!re"= J Ba$teriol 3B3A 34B2G
34C2
P!&Me;
C%emPort;
We& o( S$ien$ef
8>
Lipin"ka; B=; U/li$9; M=; )eorgopo!lo"; C= H3FF7I T%e HtrA H,egPI protein; e""ential
(or E"$%eri$%ia $oli "!r'i'al at %ig% temperat!re"; i" an enopeptia"e= J Ba$teriol
3B>A 3BF3G3BFB
P!&Me;
C%emPort;
We& o( S$ien$ef
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Ma%aEan*Miklo"; S=; Tan; M=*W=; Ra%me; L=)=; A!"!&el; #=M= H3FFFI Mole$!lar
me$%ani"m" o( &a$terial 'ir!len$e el!$iate !"ing a P"e!omona" aer!gino"aG
Caenor%a&iti" elegan" pat%ogene"i" moel= Cell FDA 2BG4D
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef
82
Martin; ,=W=; Hollo+a/; B=W=; ,ereti$; 1= H3FF8I C%ara$teri9ation o( a lo$!"
etermining t%e m!$oi "tat!" o( P"e!omona" aer!gino"a AlgU "%o+" "eO!en$e
"imilaritie" +it% a Ba$ill!" "igma (a$tor= J Ba$teriol 3B4A 3348G33D2
P!&Me;
We& o( S$ien$ef
84
Martin; ,=W=; S$%!rr; M=J=; Y!; H=; ,ereti$; 1= H3FF2I Anal/"i" o( promoter" $ontrolle
&/ t%e p!tati'e "igma (a$tor AlgU reg!lating $on'er"ion to m!$oi/ in P"e!omona"
aer!gino"aA relation"%ip to "igma E an "tre"" re"pon"e= J Ba$teriol 3BDA DDCCGDDFD
P!&Me;
We& o( S$ien$ef
8D
Mat%ee; N=; M$P%er"on; C=J=; O%man; ,=E= H3FFBI Po"ttran"lational $ontrol o( t%e algT
HalgUI*en$oe g>> (or e:pre""ion o( t%e alginate reg!lon in P"e!omona"
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Ba$teriol 3BFA 8B33G8B>7
P!&Me;
C%emPort;
We& o( S$ien$ef
8B
Mig!el; E=; Po9a*Carrion; C=; Lope9*Solanilla; E=; Ag!ilar; I=; Llama*Pala$io"; A=;
)ar$ia*Olmeo; #=; Rorig!e9*Palen9!ela; P= H>777I E'ien$e again"t a ire$t
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PlantGMi$ro&e Intera$t 38A 2>3G2>F
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef
8C
Miller; J=H= H3FB>I E:periment" in Mole$!lar )eneti$"= Col Spring Har&or; NYA Col
Spring Har&or La&orator/ Pre""
8F
Mi""iaka"; ,=; Ma/er; M=P=; Lemaire; M=; )eorgopo!lo"; C=; Raina; S= H3FFBI
Mo!lation o( t%e E"$%eri$%ia $oli "igmaE HRpoEI %eat*"%o$k tran"$ription*(a$tor
a$ti'it/ &/ t%e R"eA; R"eB an R"eC protein"= Mol Mi$ro&iol >2A 844G8B3
,ire$t LinkA
A&"tra$t
P,#H3>>DNI
27
Ne+man; M=*A=; Conra"*Stra!$%; J=; S$o5el; )=; ,aniel"; M=J=; ,o+; J=M= H3FF2I
,e(en"e*relate gene in!$tion in Bra""i$a $ampe"tri" in re"pon"e to e5ne
m!tant" o( Rant%omona" $ampe"tri" +it% altere pat%ogeni$it/= Mol PlantGMi$ro&e
Intera$t BA 448G4D8
Cro""Re(;
P!&Me;
We& o( S$ien$ef
23
Pallen; M=J= 0 Wren; B=W= H3FFBI T%e HtrA (amil/ o( "erine protea"e"= Mol Mi$ro&iol
>DA >7FG>>3
,ire$t LinkA
A&"tra$t
P,#H33>BNI
2>
PoO!et; I=; Saint; 1=; Se9ne$; E=; Simoe"; N=; Bolotin; A=; )r!""; A= H>777I HtrA i" t%e
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nat!ral protein pro$e""ing= Mol Mi$ro&iol 84A 372>G3743,OIA 37=372D6E=38D4*
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,ire$t LinkA
A&"tra$t
#!ll Arti$le HHTMLI
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Re(eren$e"
28
Ra%me; L=)=; Ste'en"; E=J=; Wol(ort; S=#=; S%ao; J=; Tompkin"; R=)=; A!"!&el; #=M=
H3FF4I Common 'ir!len$e (a$tor" (or &a$terial pat%ogeni$it/ in plant" an animal"=
S$ien$e >DCA 3CFFG3F7>
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef
22
Ra%me; L=)=; Tan; M=*W=; Le; L=; Wong; S=M=; Tompkin"; R=)=; Caler+oo; S=B=;
A!"!&el; #=M= H3FFBI U"e o( moel plant %o"t" to ienti(/ P"e!omona" aer!gino"a
'ir!len$e (a$tor"= Pro$ Natl A$a S$i USA F2A 38>24G38>47
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef
24
Rai'io; T=L= 0 Sil%a'/; T=J= H3FFFI T%e "igmaE an Cp: reg!lator/ pat%+a/"A
o'erlapping &!t i"tin$t en'elope "tre"" re"pon"e"= C!rr Opin Mi$ro&iol >A 34FG3D4
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef
2D
Roop; R=M=; II; #let$%er; T=W=; Sriranganat%an; N=M=; Bo/le; S=M=; S$%!rig; )=)= H3FF2I
Ienti5$ation o( an imm!norea$ti'e Br!$ella a&ort!" HtrA "tre"" re"pon"e protein
%omolog= In(e$t Imm!n D>A 3777G377B
P!&Me;
We& o( S$ien$ef
2B
S$%inkel; A=H=; Smit; J=J=M=; Tellingen; O=1=; BeiEnen; J=H=; Wagenaar; E=; ,eetmer; L=1= ;
et al= H3FF2I ,i"r!ption o( t%e mo!"e mr3a P*gl/$oprotein gene lea" to a
e5$ien$/ in t%e &looG&rain &arrier an to in$rea"e "en"iti'it/ to r!g"= Cell BBA
2F3G47>
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef
2C
S$%!rr; M=J=; Y!; H=; Martine9*Sala9ar; J=M=; Bo!$%er; J=C=; ,ereti$; 1= H3FFDI Control o(
AlgU; a mem&er o( t%e "igma E*like (amil/ o( "tre"" "igma (a$tor"; &/ t%e negati'e
reg!lator" M!$A an M!$B an P"e!omona" aer!gino"a $on'er"ion to m!$oi/ in
$/"ti$ 5&ro"i"= J Ba$teriol 3BCA 2FFBG4772
P!&Me;
C%emPort;
We& o( S$ien$ef
2F
S$%+ei9er; H= H3FF3I E"$%eri$%iaGP"e!omona" "%!ttle 'e$tor" eri'e (rom
pUC3C63F= )ene FBA 37FG33>
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef
47
Simp"on; J=A=; Smit%; S=E=; ,ean; R=T= H3FCFI S$a'enging &/ alginate o( (ree rai$al"
relea"e &/ ma$rop%age"= #ree Rai$al Biol Me DA 82BG848
Cro""Re(;
P!&Me;
We& o( S$ien$ef
43
Skorko*)lonek; J=; Wa+r9/no+; A=; Nr9e+"ki; N=; N!rpier9; N=; Lipin"ka; B= H3FF4I Site*
ire$te m!tagene"i" o( t%e HtrA H,egPI "erine protea"e; +%o"e proteol/ti$ a$ti'it/
i" ini"pen"a&le (or E"$%eri$%ia $oli "!r'i'al at ele'ate temperat!re"= )ene 3D8A
2BG4>,OIA 37=373D678BC*333FHF4I7727D*'
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef Time" CiteA 4>
4>
Spie""; C=; Beil; A=; E%rmann; M= H3FFFI A temperat!re*epenent "+it$% (rom
$%aperone to protea"e in a +iel/ $on"er'e %eat "%o$k protein= Cell FBA 88FG82B
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef
48
Ste'en"; E=J=; R/an; C=M=; #rie&erg; J=S=; Barn%ill; R=L=; Yarm!"%; M=L=; Tompkin"; R=)=
H3FF2I A O!antitati'e moel o( P"e!omona" aer!gino"a in(e$tion in inE!r/= J B!rn
Care Re%a&il 34A >8>G>84
Cro""Re(;
P!&Me
42
Ste'en"; L= H3FFCI Anal/"i" o( 1ir!len$e o( P"e!omona" "/ringae on Ara&iop"i"
t%aliana= P%, T%e"i"; ,epartment o( Mi$ro&iolog/ an Mole$!lar )eneti$"; Har'ar
Uni'er"it/
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Sto'er; C=N=; P%am; R=P=; Er+in; A=L=; Mi9og!$%i; S=,=; Warrener; P=; Hi$ke/; M=J= ; et
al= H>777I Complete genome "eO!en$e o( P"e!omona" aer!gino"a PAO3; an
opport!ni"ti$ pat%ogen= Nat!re 27DA F4FGFD2,OIA 37=378C6847>87BF
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef
4D
Stra!$%; N=L= 0 Be$k+it%; J= H3FCCI An E"$%eri$%ia $oli m!tation pre'enting
egraation o( a&normal peripla"mi$ protein"= Pro$ Natl A$a S$i USA C4A 34BDG
34C7
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef
4B
Stra!$%; N=L=; Jo%n"on; N=; Be$k+it%; J= H3FCFI C%ara$teri9ation o( egP; a gene
reO!ire (or proteol/"i" in t%e $ell en'elope an e""ential (or gro+t% o( E"$%eri$%ia
$oli at %ig% temperat!re= J Ba$teriol 3B3A >DCFG>DFD
P!&Me;
C%emPort;
We& o( S$ien$ef
4C
Tan; M=*W=; Ma%aEan*Miklo"; S=; A!"!&el; #=M= H3FFFaI Nilling o( Caenor%a&iti"
elegan" &/ P"e!omona" aer!gino"a !"e to moel mammalian &a$terial
pat%ogene"i"= Pro$ Natl A$a S$i USA FDA B34GB>7
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef
4F
Tan; M=*W=; Ra%me; L=)=; Sternerg; J=A=; Tompkin"; R=)=; A!"!&el; #=M= H3FFF&I
P"e!omona" aer!gino"a killing o( Caenor%a&iti" elegan" !"e to ienti(/ P=
aer!gino"a 'ir!len$e (a$tor"= Pro$ Natl A$a S$i USA FDA >27CG>238
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef
D7
Tat!m; #=M=; C%e'ille; N=#=; Mor5tt; ,= H3FF2I Cloning; $%ara$teri9ation an
$on"tr!$tion o( %trA an %trA*like m!tant" o( Br!$ella a&ort!" an t%eir "!r'i'al in
BALB*$ mi$e= Mi$ro& Pat%og 3BA >8G8D
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef
D3
T!rner; J=M= 0 Me""enger; A=J= H3FCDI O$$!rren$e; &io$%emi"tr/ an p%/"iolog/ o(
p%ena9ine pigment pro!$tion= A' Mi$ro&ial P%/"iol >BA >33G>B8
Cro""Re(;
P!&Me;
We& o( S$ien$ef
D>
Waller; P=R= 0 Sa!er; R=T= H3FFDI C%ara$teri9ation o( egP an egS; E"$%eri$%ia $oli
gene" en$oing %omolog" o( t%e ,egP protea"e= J Ba$teriol 3BCA 332DG3348
P!&Me;
C%emPort;
We& o( S$ien$ef
D8
Rie; U=,=; Her"%&erger; C=,=; S%ankar; S=; Ye; R=W=; C%akra&art/; A=M= H3FFDI Sigma
(a$torGanti*"igma (a$tor intera$tion in alginate "/nt%e"i"A in%i&ition o( AlgT &/ M!$A=
J Ba$teriol 3BCA 2FF7G2FFD
P!&Me;
C%emPort;
We& o( S$ien$ef
D2
Yamamoto; T=; Hana+a; T=; Ogata; S=; Nami/a; S= H3FFDI Ienti5$ation an
$%ara$teri9ation o( t%e Yer"inia entero$oliti$a g"rA gene; +%i$% prote$ti'el/
re"pon" to intra$ell!lar "tre"" in!$e &/ ma$rop%age p%ago$/to"i" an to
e:tra$ell!lar en'ironmental "tre""= In(e$t Imm!n D2A >FC7G>FCB
P!&Me;
C%emPort;
We& o( S$ien$ef
D4
Yamamoto; T=; Hana+a; T=; Ogata; S=; Nami/a; S= H3FFBI T%e Yer"inia entero$oliti$a
)"rA "tre"" protein; in'ol'e in intra$ell!lar "!r'i'al; i" in!$e &/ ma$rop%age
p%ago$/to"i"= In(e$t Imm!n D4A >3F7G>3FD
P!&Me;
C%emPort;
We& o( S$ien$ef
DD
Yani"$%*Perron; C=; 1ieira; J=; Me""ing; J= H3FC4I Impro'e M38 p%age $loning 'e$tor"
an %o"t "train"A n!$leotie "eO!en$e" o( t%e M38mp3C an pUC3F 'e$tor"= )ene
88A 378G33F
Cro""Re(;
P!&Me;
C%emPort;
We& o( S$ien$ef Time" CiteA 38C78
DB
Y!; H=; S$%!rr; M=J=; ,ereti$; 1= H3FF4I #!n$tional eO!i'alen$e o( E"$%eri$%ia $oli
"igma E an P"e!omona" aer!gino"a AlgUA E= $oli rpoE re"tore" m!$oi/ an
re!$e" "en"iti'it/ to rea$ti'e o:/gen intermeiate" in algU m!tant" o( P=
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P!&Me;
We& o( S$ien$ef
DC
Y!; H=; Bo!$%er; J=C=; Hi&ler; N=S=; ,ereti$; 1= H3FFDI 1ir!len$e propertie" o(
P"e!omona" aer!gino"a la$king t%e e:treme*"tre"" "igma (a$tor AlgU H"igmaEI=
In(e$t Imm!n D2A >BB2G>BC3
P!&Me;
C%emPort;
We& o( S$ien$ef
DF
Y!; H=; Hane"; M=; C%ri"p; C=E=; Bo!$%er; J=C=; ,ereti$; 1= H3FFCI Mi$ro&ial
pat%ogene"i" in $/"ti$ 5&ro"i"A p!lmonar/ $learan$e o( m!$oi P"e!omona"
aer!gino"a an inSammation in a mo!"e moel o( repeate re"pirator/ $%allenge=
In(e$t Imm!n DDA >C7G>CC
P!&Me;
We& o( S$ien$ef
B7
Y!; J=; Penalo9a*1a9O!e9; A=; C%akra&art/; A=M=; Bener; C=L= H3FFFI In'ol'ement o(
t%e e:opol/"a$$%arie alginate in t%e 'ir!len$e an epip%/ti$ 5tne"" o(
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