REPORT
Inc eased sensi i i y o p o ein syn hesis
inhibi o s in cells lacking mRNA
JESÚS DE LA CRUZ
1,2
and AGUSTÍN VIOQUE
1
1Ins i u o de Bioquímica Vege al y Fo osín esis,Uni e sidad de Se illa-CSIC,
Amé ico Vespucio s/n,E-41092 Se illa,Spain
ABSTRACT
mRNA (also known as Ss A o 10Sa RNA) is in ol ed in a
ans
- ansla ion eac ion ha con ibu es o he ecycling
o s alled ibosomes a he 39end o an mRNA lacking a s op codon o a an in e nal mRNA clus e o a e codons.
Inac i a ion o he
ss A
gene in mos bac e ia esul s in iable cells bea ing sub le pheno ypes, such as empe a u e-
sensi i e g ow h. He ein, we epo on he unc ional cha ac e iza ion o he
ss A
gene in he cyanobac e ium
Syn-
echocys is
sp. s ain PCC6803. Dele ion o he
ss A
gene in
Synechocys is
esul ed in iable cells wi h a g ow h a e
iden ical o wild- ype cells. Howe e , null
ss A
cells (
D
ss A
) we e no iable in he p esence o he p o ein syn hesis
inhibi o s chlo amphenicol, lincomycin, spi amycin, ylosin, e y h omycin, and spec inomycin a low doses ha do
no signi ican ly a ec he g ow h o wild- ype cells. Sensi i i y o
D
ss A
cells simila o wild- ype cells was obse ed
wi h kasugamycin, usidic acid, hios ep on, and pu omycin. An ibio ics un ela ed o p o ein syn hesis, such as
ampicillin o i ampicin, had no di e en ial e ec on he
D
ss A
s ain. Fu he mo e, dele ion o he
ss A
gene is
su icien o impai global p o ein syn hesis when chlo amphenicol is added a suble hal concen a ions o he
wild- ype s ain. These esul s indica e ha ibosomes s alled by some p o ein syn hesis inhibi o s can be ecycled
by mRNA. In addi ion, his sugges s ha he i s elonga ion cycle wi h mRNA, which inco po a es a noncoded
alanine on he g owing pep ide chain, may ha e mechanis ic di e ences wi h he no mal elonga ion cycles ha
bypasses he block p oduced by hese speci ic an ibio ics. mRNA inac i a ion could be an use ul he apeu ic a ge
o inc ease he sensi i i y o pa hogenic bac e ia agains an ibio ics.
Keywo ds: an ibio ics;
ss A
;
Synechocys is
; mRNA; ansla ion
INTRODUCTION
Bac e ial cells con ain a panoply o s able non ans-
la ed RNAs such as ibosomal and ans e RNAs
( RNAs and RNAs; e iewed in Wassa man e al+,
1999)+Amongs hese RNAs, he so-called mRNA(also
known as Ss Ao 10Sa RNA) was one o he i s o be
iden i ied in
Esche ichia coli
(Jain e al+, 1982);how-
e e ,i s unc ion has emained elusi e o a decade
The mRNA is encoded by he
ss A
gene,which is
highlyconse edinalleubac e ia(Knudsen e al+, 2001)+
In i o, mRNA is igh ly associa ed o he SmpB p o-
ein and less igh ly o o he p o eins (Ka zai e al+,
1999;Ka zai & Saue ,2001)+Di e en e idence indi-
ca es ha mRNA plays a ole in ansla ion ( o a e-
iew,see Mu o e al+, 1998;Ka zai e al+, 2000)+ mRNA
can o m in i o a RNA hal molecule consis ing o a
amino acid accep o a m and a TCC s em-loop (Ko-
mine e al+, 1994)+The p esence o his RNA-like s uc-
u e is u he suppo ed by he ac ha he p ecu so
o mRNA(p e- mRNA),as all p e- RNAs,is 59-end p o-
cessed by RNase P,and mRNA is aminoacyla ed by
alanyl- RNA syn he ase (Komine e al+, 1994)+In addi-
ion, healanyla ed- mRNAin e ac swi h elonga ion ac-
o Tu (EF-Tu) and GTP o ming a e na y complex
(Rudinge -Thi ion e al+, 1999) and is ound associ-
a ed wi h 70S ibosomes (Tadaki e al+, 1996)+Finally,
mRNAs ha e a sho eading ame coding o abou
11 amino acids (Knudsen e al+, 2001),which a e added
o he C- e minuso an incomple enascen p o ein ans-
la ed om unca ed mRNAs (Keile e al+, 1996)+
Based on some o hese obse a ions,Saue and
cowo ke s p oposed he “
ans
- ansla ion” model o
he biological unc ion o mRNA (Keile e al+, 1996)+
Rep in eques s o:Agus ín Vioque,Ins i u o de Bioquímica Veg-
e al y Fo osín esis,Uni e sidad de Se illa-CSIC,Cen o de In es i-
gaciones Cien í icas Isla de la Ca uja,A da+Amé ico Vespucio s/n,
E-41092 Se illa,Spain;e-mail: ioque@us+es+
2P esen add ess:Depa amen o de Gené ica,Facul ad de Bio-
logía,Uni e sidad de Se illa,Reina Me cedes 6,E-41012 Se illa,
Spain+
RNA
(2001),
7
:1708–1716+Camb idge Uni e si y P ess+P in ed in he USA+
Copy igh © 2001 RNA Socie y+
DOI:10+1017+S1355838201011281
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Acco ding o his model,alanine-cha ged mRNAasso-
cia ed wi h EF-Tu en e s heAsi e o a ibosome s alled
a he 39end o a unca ed mRNA lacking a s op co-
don+Then, he noncoded alanine is inco po a ed o he
nascen polypep ide chain h ough a cycle o ansla-
ionelonga ion,be o e he mRNA-like domain o mRNA
eplaces he unca ed mRNA on he ibosome+Then,
his domain is used as a empla e o add a sho pep-
ide o he nascen polypep ide be o e ansla ion e -
mina es and a agged p o ein is eleased ( o de ails,
see Keile e al+, 1996;Mu o e al+, 1998)+The sho
pep ide ac s as a ecogni ion sequence ha di ec s he
p o ein o quick deg ada ion by speci ic p o eases
(Go esman e al+, 1998;Le chenko e al+, 2000)+The e-
o e, mRNA has a double unc ion:Fi s , ibosomes
ha a e s alled a unca ed mRNAs lacking a e mina-
ion codon a e eleased+Second, he p ema u ely
unca ed p o eins a e apidly deg aded,a oiding ac-
cumula ion+Bo h unc ion ha e been sepa a ed gene -
ically by enginee ing cells ha ca y a
ss A
gene coding
o an al e ed ag ha is esis an o p o eases,o al-
e na i ely, ha canno be aminoacyla ed (Keile e al+,
1996;Wi hey & F iedman,1999;Huang e al+, 2000)+
F om hese s udies,i seems clea ha ibosome e-
lease and no p o ein agging is he ele an unc ion o
mRNA (Keile e al+, 1996;Wi hey & F iedman,1999;
Huang e al+, 2000)+In addi ion,i has been demon-
s a ed ha mRNA-media ed
ans
- ansla ion occu s
qui e equen ly in no mally g owing cells and no only
a unca ed mRNAs lacking a e mina ion codon (Abo
e al+, 2000)+Tagging by mRNA occu s a a e codons
in an mRNA o e en a he na u al e mina ion codon
(Roche & Saue ,1999,2001)+This sugges s ha ibo-
somepausingo s alling byanumbe o di e en causes
can ac i a e mRNA unc ion+Howe e ,se e al ques-
ions emain unsol ed:How does mRNA ecognize
s alled ibosomes,wha is he mechanism o inco po-
a ion o he noncoded alanine,and how does he ibo-
some esume in- ame ansla ion a he mRNA-like
domain o mRNA?
Mo eo e ,despi e i s impo an biological unc ion and
i s ubiqui ous p esence in he eubac e ial kingdom, he
p ecise physiological ole o mRNA s ill emains elu-
si e,as i is,in gene al,dispensable o cell iabili y and
i has been p o ed o be essen ial only in
Neisse ia
gono hoeae
and
Mycoplasma
(Hu chison e al+, 1999;
Huang e al+, 2000)+Howe e ,cells lacking a unc ional
mRNA exhibi a a ie y o sub le pheno ypes such as
slow g ow h a high empe a u e o unde di e en
s esses, educed mo ili y,inhibi ion o phage g ow h,
an inc ease in he ac i i y o an uniden i ied p o ease,
enhanced ac i i y o se e al ep esso s o educed
pa hogenesis (Ka zai e al+, 2000;Mu o e al+, 2000;
and e e ences he ein)+In e es ingly,Sugi a and co-
wo ke s could no inac i a e he
ss A
gene in he cy-
anobac e ium
Synechococcus
sp+s ain PCC6301,
sugges ing an essen ial unc ion o his gene (Wa-
anabe e al+, 1998)+Fo his eason,we unde ook he
unc ional cha ac e iza ion o he
ss A
gene in he cy-
anobac e ium
Synechocys is
sp+s ain PCC6803,in
he con ex o an ongoing p ojec on he analysis o
s able RNAs om his cyanobac e ium+He ein,we e-
po ha
Synechocys is
cellsdis up ed o he
ss A
gene
(D
ss A
cells) show no g ow h de ec pheno ype unde
no mal labo a o y condi ions+Howe e ,aD
ss A
s ain
is no iable when cul i a ed a low concen a ions o
se e al p o ein syn hesis inhibi o s+In addi ion,global
p o ein syn hesis is impai ed a low concen a ion o
chlo amphenicol in a D
ss A
s ain bu no in a wild- ype
s ain+We conclude ha mRNA has a ole in ecycling
s alled ibosomes when hey a e blocked by hose p o-
ein syn hesis inhibi o s+We assume ha he i s elon-
ga ion cycle ha inco po a es he noncoded alanine o
nascen polypep ide chains is esis an o he bound
an ibio ic+
RESULTS AND DISCUSSION
mRNA is dispensable o g ow h
To s udy he cellula unc ion o
Synechocys is
mRNA,
we i s dis up ed he
ss A
gene by eplacing mos o
he gene sequence wi h a kanamycin esis ance (Km
)
casse e (Fig+1A)+Al hough he
ss A
gene is no pa o
a polycis onic uni (Kaneko e al+, 1996), he casse e
was inse ed in bo h o ien a ions o con ol o hypo-
he ical pola e ec s+The dis up ed copy o
ss A
could
be comple ely seg ega ed as es ed by sou he n blo
(da a no shown)+The e o e,cells lacking a unc ional
ss A
gene seem o be iable+
To u he con i m he nonessen ial ole o mRNA,
we de e mined i s s eady-s a e le els by no he n blo
and p ime ex ension analyses in wild- ype and D
ss A
s ains+As seen in Figu e 1B,C,dis up ion o he
ss A
gene esul ed in he loss o ma u e mRNA+In addi ion,
we de ec ed an addi ional band in he wild- ype s ain
by p ime ex ension analysis (Fig+1C,lane 1),which
we a ibu e o he p ecu so o mRNA (Tous e al+,
2001)+Al oge he , hese expe imen s indica e ha
ss A
is a dispensable gene in
Synechocys is
,in disag ee-
men wi h he da a om Sugi a and cowo ke s o
Syn-
echococcus
(Wa anabe e al+, 1998)+
Dis up ion o
ss A
causes
no appa en g ow h de ec
To unde s and he nonessen ial ole o mRNA, he
g ow h a e o he D
ss A
s ain was compa ed o ha o
he wild- ype s ain+As seen in Figu e 2A,when cells
we e cul i a ed in ni a e con aining BG-11 medium a
308C (see Ma e ials and Me hods), he g ow h a es
we e iden ical o wild- ype and D
ss A
s ains+In addi-
ion,D
ss A
cells we e as mo ile as wild- ype cells and
we could no de ec any mo phological di e ence when
mRNA and ansla ion inhibi o s
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cells we e obse ed unde he ligh mic oscope (da a
no shown)+We conclude ha
Synechocys is
mRNAis
o ally dispensable unde no mal labo a o y g ow h
condi ions+
mRNA equi emen unde se e al s esses:
Sensi i i y o p o ein syn hesis inhibi o s
To es ima e di e ences in g ow h a es unde di e en
s ess ulcondi ions,se ialdilu ions o cul u es omwild-
ype and D
ss A
cells we e spo ed on BG11 medium
pla es,and pla es we e incuba ed a 308C (see Ma e-
ials and Me hods)+S ess ul condi ions we e:expo-
su e o UV i adia ion (2,000 KJ{s
21
) o 2 o10min,
DTT (250 mM),2-me cap oe hanol (5 mM), ea men
a 258C,378C,and 458C o 30 min+In all condi ions,
no di e ences in he su i al o he g ow h a es o he
wild- ypeand D
ss A
s ainswe e ound(da a no shown)+
Because mRNA plays a ole on ansla ion (see
In oduc ion),we also measu ed g ow h a es o bo h
s ains in he p esence o low concen a ions o an i-
bio ics a ec ing p o ein syn hesis+As seen in Table 1
and Figu e 2B, he D
ss A
s ains do no g ow in he
p esence o suble hal concen a ions o he wild- ype
s ain o chlo amphenicol,e y h omycin,lincomycin,
spec inomycin,spi amycin,and ylosin+These da a in-
dica e ha he su i al a e o he wild- ype s ain is
10- o 1000- old highe han ha o he D
ss A
s ains
(Fig+2B)+In e es ingly,all hese d ugs excep spec i-
nomycin ha e been desc ibed o bind o he pep idyl
ans e ase cen e ,which has been localized wi hin
domain V o he 23S RNA (Gale e al+, 1981;
Rod íguez-Fonseca e al+, 1995;Nissen e al+, 2000;
Poulsen e al+, 2000)+Fu he mo e,all hese d ugs
excep e y h omycin and spec inomycin inhibi he pep-
idyl ans e ase eac ion (Gale e al+, 1981)+Re-
FIGURE 1. Dis up ion o he
Synechocys is ss A
gene+A: Schema ic ep esen a ion o he
Synechocys is
genomic egion
whe e he
ss A
gene is loca ed+A
Hin
cII Km casse e (1+3 kb) con aining he neomycin phospho ans e ase gene (
np
) was
inse ed be ween he blun -ended
Hin
dIII and
Cla
I si es in bo h o ien a ions (D
ss A-1
and D
ss A-2
)+B: No he n blo
analysis; o al RNA was ex ac ed om wild- ype and null
ss A
(D
ss A-1
and D
ss A-2
) cells and hyb idyzed wi h a
Sma
I-
Nco
I agmen con aining he
ss A
gene (uppe panel) o a
npB
gene p obe (bo om panel;Vioque,1992)+C: P ime
ex ension using he oligonucleo ide 10SAPE (see Ma e ials and Me hods) on o al RNA ex ac ed om wild- ype and
null
ss A
(D
ss A-1
and D
ss A-2
) cells+A ows poin s o he ex ension p oduc s iden i ying he pu a i e ansc ip ion s a si e
(p e- mRNA) and he 59end o ma u e mRNA ( mRNA) only in wild- ype cells+Fo he p ime ex ension shown in lanes 3
and 4,a 10- old mola excess o cold p ime was mixed wi h he adiolabeled p ime be o e annealing+Asequencing eac ion
on he wild- ype
ss A
gene using he same p ime is also shown+
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cen ly,i has been shown ha e y h omycin binds o
he polypep ide unnel,sugges ing a simple s e ic-
hind ance mechanism o inhibi ion o elonga ion
(Gabash ili e al+, 2001)+Spec inomycin also inhibi s
ansloca ion,bu i binds o speci ic si es on 16S RNA
(Bilgin e al+, 1990;Ca e e al+, 2000)+
FIGURE 2. mRNAis equi ed o su i al in he p esence o p o ein syn hesis inhibi o s+A: G ow h compa ison o wild- ype
and D
ss A
s ains+Cul u es we e inocula ed a an ini ial concen a ion o 0+5mg{mL21chlo ophyll+Cells we e g own on
ni a e con aining BG11 medium o up o 12 days and g ow h a e was measu ed as he concen a ion o chlo ophyll pe
millili e o cul u e+Wild- ype (d),D
ss A-1
(n) and D
ss A-2
(,) s ains+B: Cells om wild- ype (w ),null
ss A
(D
ss A-1
and
D
ss A-2
) and null
c O
(D
c O
) s ains we e g own o 3 OD750 uni s+Se ial dilu ions (dilu ion ac o 1 o 103) we e spo ed
on o ni a e con aining BG11 medium pla es a he indica ed concen a ions o an ibio ics+Pla es we e incuba ed as
desc ibed in Ma e ials and Me hods o 7 days+Con ol:no an ibio ic added;Cm:chlo amphenicol;E :e y h omycin;Lc:
lincomycin;Sp:spec inomycin;Sy:spi amycin;Ty: ylosin+
mRNA and ansla ion inhibi o s
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As also shown in Table 1,an ibio ics a ec ing ans-
la ion ini ia ion o EF-Tu and EF-G-media ed s eps
du ing ansloca ion (excep e y h omycin and spec i-
nomycin) do no di e en ially a ec he D
ss A
s ain as
compa ed o he wild- ype s ain+In addi ion, he D
ss A
s ains g ow be e han he wild- ype s ain in he p es-
ence o any concen a ion o kanamycin,neomycin,o
s ep omycin+This g ow h ad an age is due o he unc-
ion o he neomycin phospho ans e ase gene p esen
in he Km
casse e in oduced du ing he ans o ma-
ion o he D
ss A
cells+This is demons a ed by he
con ol D
c O
s ain (Fe nández-González e al+, 1997),
which is a Km
mu an ha p ese es a unc ional
ss A
gene+As expec ed, he D
c O
s ain showed also a
g ow had an ageonpla escon ainingkanamycin,neo-
mycin,o s ep omycin (da a no shown)+Finally,D
ss A
and wild- ype cells a e equally a ec ed by o he an i-
bio ics ha do no in e e e wi h p o ein syn hesis such
as ampicillin o i ampicin (Table 1)+We conclude ha
mRNA is essen ial o g ow h in he p esence o low
concen a ions o chlo amphenicol,lincomycin,spi a-
mycin, ylosin,e y h omycin,and spec inomycin+
P o ein syn hesis is impai ed in
D
ss A
cells p e ea ed wi h a
low dose o chlo amphenicol
To be e de ine he al e ed esis ance o D
ss A
cells o
he d ugs men ioned in he p e ious sec ion,we ana-
lyzed he wild- ype and D
ss A
s ains o p o ein syn-
hesis a e by in i o inco po a ion o [
35
S]me hionine
(see Ma e ials and Me hods)+As shown in Figu e 3A,
when no an ibio ic was added, he ela i e amoun o
[
35
S]me hionine inco po a ed in o p o eins was ep o-
ducibly simila in bo h wild- ype and D
ss A
s ains+
Howe e ,when cells we e ea edwi h 1 mg{mL
21
chlo -
amphenicol o 30 min,mo e han a 50% educ ion in
[
35
S]me hionine inco po a ion in o p o eins was ob-
se ed in D
ss A
cells when compa ed o he wild- ype
s ain (Fig+3A)+Fu he mo e,we could no de ec any
signi ica i e e ec on he [
35
S]me hionine up ake (da a
no shown)+To in es iga e i he ansla ion a e o a se
o p o eins is mo e a ec ed han o he ones by chlo -
amphenicol,we analyzed SDS/PAGE p o iles o
35
S-
labeled p o eins om bo h wild- ype and D
ss A
cells
ea ed o no wi h he d ug+As shown in Figu e 3B,no
di e enceswe e oundalong he p o iles (compa e lines
1 and 3 wi h 2 and 4)+Al oge he , hese esul s indica e
ha mRNA is equi ed o op imal p o ein syn hesis in
he p esence o suble hal concen a ions o chlo am-
phenicol+This equi emen is no speci ic o a se o
p o eins, he e o e s ongly sugges ing ha global im-
pai men o p o ein syn hesis is he p ima y cause o
he le hali y o he D
ss A
s ains when g own in he
p esence o low concen a ions o p o ein syn hesis
inhibi o s+
CONCLUSIONS
In his wo k,we ha e demons a ed ha mRNA is es-
sen ial o su i al in he p esence o suble hal con-
cen a ions o se e al p o ein syn hesis inhibi o s+In
hese condi ions,only a ac ion o ibosomes would
bind he an ibio ic and becomes blocked on a mRNA+
Assuming ha hese blocked ibosomes can ec ui
cha ged mRNA and mo eo e ,assuming ha he i s
elonga ion cycle ha inco po a es he noncoded ala-
nine is esis an o he bound an ibio ic,which mus be
displaced, hen ibosomes ha e he oppo uni y o be
eleased+This abili y is no p esen in D
ss A
cells; he e-
o e,blocked ibosomes accumula e, ansla ion is im-
pai ed,and cells do no g ow+
mRNA-media ed agginghasbeen desc ibed in h ee
ci cums ances:(1) when ibosomes each he end o a
unca ed mRNA (Keile e al+, 1996),(2) when ibo-
somes s all a a e codons (Roche & Saue ,1999),and
(3) a na u al s op codons (Roche & Saue ,2001)+
In hese cases, he ibosomal A-si e is emp y+Ou e-
sul s p o ide e idence ha ibosomes s alled by some
p o ein syn hesis inhibi o s can also be a subs a e o
mRNA+Howe e ,in he p esence o pep idyl ans e -
ase inhibi o s, he ibosomalA-si e can be occupied by
he co ec aminoacyl- RNAo he mRNA+In he p es-
ence o e y h omycin and spec inomycin, he ac ion o
mRNAon s alled ibosomes is mo e di icul o explain
because pep idyl- RNA is in he ibosomal A-si e+In
TABLE 1+E ec o di e en an ibio ics on he g ow h a e o wild-
ype and D
ss A
cells om
Synechocys is
sp+PCC6803+
Inhibi o Func ion a ec ed G ow h
di e ence
a
Ampicillin Cell wall syn hesis None
Ri ampicin T ansc ip ion None
Kasugamycin Ini ia ion ( Me - RNA binding) None
Kanamycin T ansla ion accu acy 1
Neomycin T ansla ion accu acy 1
S ep omycin T ansla ion accu acy 1
Te acycline aa- RNA{EF-Tu{GTP binding None
Chlo amphenicol Pep ide bond o ma ion 2
Lincomycin Pep ide bond o ma ion 2
Spi amycin Pep ide bond o ma ion 2
Tylosin Pep ide bond o ma ion 2
E y h omycin Elonga ion 2
Spec inomycin T ansloca ion (EF-G{GTP in e ac ion) 2
Fusidic acid T ansloca ion (EF-G{GDP elease) None
Thios ep on T ansloca ion (EF-G{GDP u no e ) None
Pu omycin Elonga ion None
Rela i e g ow h a e was compa ed by do ing se ial dilu ions o expo-
nen ially g owing cells on pla es con aining he indica ed an ibio ics a
suble hal concen a ions o he wild- ype s ain (see Ma e ials and
Me hods)+
a
None:simila g ow h o wild- ype and D
ss A
s ains;1:be e g ow h
o he D
ss A
s ain;2:be e g ow h o he wild- ype s ain+No e ha he
be e g ow h o he D
ss A
s ains in kanamycin,neomycin,and s ep o-
mycin is no ela ed o he loss o mRNA unc ion bu o he in oduced
Km
casse e ollowing ans o ma ion+
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any case,ou da a s ongly sugges ha mRNA pa -
icipa es in a p oduc i e elonga ion cycle in he p es-
ence o any o he abo e men ioned inhibi o s,mos
likely by displacing he an ibio ic+P o ein syn hesis an i-
bio ics had been and s ill a e ex emely use ul ools in
he dissec ion o he complex mechanisms o ansla-
ion+Fu he in i o and in i o expe imen s using hese
an ibio ics may be e y use ul o elucida e he mech-
anism o mRNA-media ed
ans
- ansla ion+
Finally, he e is a g ea in e es in he de elopmen o
new oolsagains pa hogenicmic oo ganisms+Al hough
mRNA is no essen ial o g ow h,ou esul s indica e
ha inhibi ion o mRNAac i i ycould inc ease hesensi-
i i y o ansla ion an ibio ics o clinical ele ance+I
ou esul s can be gene alized o pa hogenic bac e ia,
mRNA could be an a ac i e a ge o he de elop-
men o new speci ic d ugs,as mRNAis no p esen in
animal cells+Thus,a combined deli e y o a mRNAin-
hibi o andadocumen edan ibio ica alowe dosewould
be mo e e ec i e agains pa hogenic mic oo ganisms+
MATERIALS AND METHODS
S ains, media, and g ow h condi ions
Synechocys is
sp+s ain PCC6803 was g own in ni a e con-
aining BG-11 medium (Rippka e al+, 1979)+Fo pla es, he
medium was solidi ied wi h 1% (w/ ),sepa a ely au ocla ed
aga (Di co)+Cul u es we e g own a 308C in he ligh (50–
100 meins eins{m
22
{s
21
;whi e ligh luo escen lamps),wi h
shaking (80–100 pm) o liquid cul u es+G ow h was moni-
o ed by OD
750
o chlo ophyll con en ha was de e mined as
FIGURE 3. P o ein syn hesis is impai ed in D
ss A
cells+A: P o ein syn hesis a es in wild- ype (w ) and D
ss A
cells+
Cul u es (3 OD750 uni s) we e incuba ed in he absence o p esence o chlo amphenicol (1 mg{mL21) o 30 min+[35S]me-
hionine was added ei he immedia ely (0 min,uppe panel) o 30 min a e he addi ion o chlo amphenicol (30 min,uppe
panel)+Incuba ion wi h [35S]me hionine was o 15 min+The adioac i i y p esen in he TCA-insoluble ma e ial was coun ed
and di ided by ha aken up in o cells+The ansla ion a e o each s ain is he a e age o a leas h ee independen
expe imen s and he s anda d de ia ions a e gi en as ba s+B: De ec ion o 35S-labeled p o eins in wild- ype and D
ss A
cells+
Cul u es (3 OD750 uni s) we e incuba ed in he absence o p esence o chlo amphenicol (1 mg{mL21) o 30 min and hen
wi h [35S]me hionine o 15 min+One OD750 uni was lysed by hea ing a 608C in Laemmli bu e +Equal amoun s o
adioac i i y (20,000 cpm) we e subjec ed o SDS/PAGE and 35S-labeled p o eins we e de ec ed by au o adiog aphy+Size
ma ke s a e indica ed on he le +
mRNA and ansla ion inhibi o s
1713
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p e iouslydesc ibed(Mackinney,1941)+An ibio ic pla es we e
p epa ed by adding he d ugs om s ock solu ions in o he
medium be o e pou ing he pla es+An ibio ics we e used a a
ange o concen a ions be ween 1 ng{mL
21
and he con-
cen a ion a which wild- ype cells die+The ollowing an i-
bio ics we e used:ampicillin (sodium sal ),chlo amphenicol,
e y h omycin, usidic acid (sodium sal ),kanamycin sul a e,
kasugamycin,lincomycin hyd ochlo ide,neomycin,pu omy-
cindihyd ochlo ide, i ampicin,spec inomycindihyd ochlo ide,
spi amycin,s ep omycin sul a e, e acycline hyd ochlo ide,
hios ep on,and ylosin a a e+All an ibio ics excep kasuga-
mycin we e o analy ical g ade+Kasugamycin was a gene -
ous gi om D +A+Jiménez+
Synechocys is
D
c O
s ain is a
Km
dis up an o he b-ca o ene ke olase gene (Fe nández-
González e al+, 1997)
. Esche ichia coli
DH5awas used o
ecombinan DNA echniques and g own in Lu ia–Be ani me-
dium wi h o wi hou an app op ia e an ibio ic+
DNA manipula ion
All ecombinan DNAexpe imen s we e pe o med acco ding
o s anda d p ocedu es (Samb ook e al+, 1989)+Sou he n
blo ing was done as p e iously desc ibed (Samb ook e al+,
1989)+To clone he
ss A
gene, wo p ime s,based on he
published coo dina es om he
Synechocys is
sp+PCC6803
ch omosome sequence (Kaneko e al+, 1996),we e syn-
hesized:10SAF,59-CGCTTGTGGATCCTGTCCCAG-39(a
Bam
HI si e is unde lined) and 10SAR,59-TAACCGCTCGAG
TAAAGTACTGTTACTGG-39(ch omosome homology e-
gion is in bold ype and a
Xho
I si e is unde lined)+PCRs we e
pe o med by a s anda d p ocedu e (Samb ook e al+, 1989)+
The PCR p oduc was cloned as a 1+5-kb
Bam
HI-
Xho
I ag-
men in opBluesc ip KS(1)(S a agene)diges ed wi h
Bam
HI
and
Xho
I o gene a e pBS-ss A+
Inac i a ion o
ss A
in
Synechocys is
cells
To inac i a e he
ss A
gene,pBS-ss A was diges ed wi h
Hin
dIII and
Cla
I, hen ea ed wi h Klenow DNA polyme ase
and dephospho yla ed+The esul ing blun -ended open plas-
mid lacking mos o he
ss A
gene sequence was liga ed o a
1+3-kb
Hin
cII agmen con aining a Km
casse e (C+K1) om
Tn5 (Elhai & Wolk,1988)+Two plasmids con aining he cas-
se e in di e en o ien a ion (pBS-D
ss A-1
and pBS-D
ss A-2
,
espec i ely) we e chosen o
Synechocys is
ans o ma ion+
Cells we e ans o med using he me hod om Chau a e al+
(1986),excep ha cells we e sp ead ou on o ni ocellulose
il e s (Nucleopo e REC-85,Wha man)+Km
ans o man s
we e selec ed on 50 mg{mL
21
Km-con aining BG-11 pla es+
To acili a e seg ega ion o he mu an ch omosomes, he Km
ans o man s we e g own o h ee ounds on 100,200,and
300 mg{mL
21
Km-con aining BG-11 pla es+Co ec in eg a-
ion and o al seg ega ion o he mu an s ains we e checked
by sou he n blo analysis+Two seg egan s ha bo ing he Km
casse e in di e en o ien a ions (D
ss A-1
and D
ss A-2
) we e
chosen o u he expe imen s+
RNA analyses
To al
Synechocys is
RNAwas isola ed om liquid cul u es as
p e iously desc ibed (Na a o & Flo encio,1996)+Dena u ed
RNA(5 mg) wassize ac iona edon7%(w/ )polyac ylamide-
8 M u ea gels+RNA was ans e ed o and immobilized on
Hybond N
1
nylon memb anes (Ame sham-Pha macia) as
p e iously desc ibed (K essle e al+, 1997)+P obes we e la-
beled wi h a DNA labeling ki (Ready o Go,Ame sham-
Pha macia)+P ehyb idiza ion and hyb idiza ion we e done as
p e iously desc ibed (K essle e al+, 1997)+
P ime ex ension was done on he same RNA samples
ha we e used o no he n analysis acco ding o Ga cía-
Domínguez e al+(2000)+Oligonucleo ide 10SAPE (59-CTA
GGCTGCTATGGCTACC-39) was used as a p ime +To iden i y
he posi ions o he p ime ex ension s op,plasmid-bo ne
ss A
was sequenced wi h he same p ime +AMV e e se
ansc ip aseandRNAgua dwe e pu chased omAme sham-
Pha macia+
Chlo amphenicol inhibi ion
o p o ein syn hesis
A modi ied p ocedu e om Xu e al+(2000) was used o de-
e mine p o ein syn hesis a es;2mL o chlo amphenicol dis-
sol ed in e hanol (1 mg{mL
21
inal concen a ion) o 2 mLo
e hanol we e added o 1-mL cul u es o
Synechocys is
wild-
ype and D
ss A-1
s ain (3 OD
750
uni s) and incuba ed o
di e en imes unde he g ow h condi ions desc ibed abo e+
Labeling was done using 10 mCi L-[
35
S]me hionine (1,000
Ci/mmol) o 15 min+Then,hal o each sample was added o
0+5 mL ice-cold BG11 medium plus 1 mg{mL
21
me hionine,
il e ed on o a Wha man GF/C pape ,and washed h ee imes
wi h 5 mL BG11 medium; he il e was inally imme sed in
scin illa ion cock ail (5up ake sample)+The emaining hal o
each sample was incuba ed wi h TCA and SDS [ inal con-
cen a ions:10% (w/ ) TCA,1% (w/ ) SDS] o 1ha 48C,
hen il e ed on o Wha man GF/C pape and washed h ee
imes wi h 5 mL 10% (w/ ) TCA, ollowed by an addi ional
wash wi h 5 mL ace one+The il e was inally imme sed in
scin illa ion cock ail (5inco po a ion sample)+The adioac i -
i y o he samples was measu ed by coun ing and quench
co ec ed+The p o ein syn hesis a e was exp essed as he
inco po a iondi idedby he up ake ocompensa e o changes
in label up ake among he samples+
P o ein analyses
P o einconcen a ionwasde e minedby heB ad o dme hod
using o oalbumin as a s anda d (B ad o d,1976)+P o ein cell
ex ac s we e p epa ed by adding Laemmli bu e o 3 OD
750
uni s o cells and hea ing o 15 min a 608C (Ausubel e al+,
1994)+In i o p o ein labeling expe imen s we e pe o med
as desc ibed abo e using cul u es o
Synechocys is
wild-
ype and D
ss A-1
s ains+A 3OD
750
uni s, he cul u es we e
di ided in wo hal es;one was incuba ed o a leas 10 min
wi h 2 mLo chlo amphenicol dissol ed in e hanol (1 mg{mL
21
inal concen a ion) and he o he was ea ed wi h 2 mL e h-
anol o he same pe iod o ime+Then,1 mLo each hal was
incuba ed wi h 10 mCi L-[
35
S]me hionine (1,000 Ci/mmol) o
15 min+Cells we e washed wice by cen i uga ion wi h 1 mL
o BG11 medium supplemen ed wi h 1 mg{mL
21
me hionine
and esuspended in 50 mL o Laemmli bu e +Labeled p o-
eins we e analyzed by SDS/PAGE (equal amoun s o adio-
ac i i y was loaded in each well) ollowed by au o adiog aphy+
1714
J. de la C uz and A. Vioque
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ACKNOWLEDGMENTS
We hanks D +A+Jiménez (CBM,Mad id) o p o iding ka-
sugamycin,L+López-Mau y o help du ing he p epa a ion o
Figu e 2,D +M+A+Vega-Palas o ui ul discussions,and D +
D+K essle (Biozen um,Basel) o c i ical eading o hemanu-
sc ip +J+d+l+C+acknowledges inancial suppo om he Span-
ish Go e nmen (Con a o de Reinco po ación,Minis e io de
Educación y Cul u a)+This wo k was suppo ed by g an s
om he Human F on ie Science O ganiza ion (RG291/
1997),Di ección Gene al de Enseñanza Supe io (PB97-
0732),and Jun a de Andalucía (CVI215)+
Recei ed July 19, 2001; e u ned o e ision
Augus 5, 2001; e ised manusc ip ecei ed
Sep embe 21, 2001
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