Vol.
176,
No.
20
JOURNAL
OF
BACTERIOLOGY,
Oc .
1994,
p.
6397-6401
0021-9193/94/$04.00+0
Copy igh
©
1994,
Ame ican
Socie y
o
Mic obiology
Analysis
o
Exp ession
o
he
a gC
and
a gD
Genes
in
he
Cyanobac e ium
Anabaena
sp.
S ain
PCC
7120
BELEN
FLORIANO,
ANTONIA
HERRERO,
AND
ENRIQUE
FLORES*
Ins i u o
de
Bioquimica
Vege al
y
Fo osin esis,
Uni e sidad
de
Se illa-Consejo
Supe io
de
In es igaciones
Scien i icas,
E-41080
Se illa,
Spain
Recei ed
14
June
1994/Accep ed
8
Augus
1994
A
cloned
DNA
agmen
om
Anabaena
sp.
s ain
PCC
7120
ha
complemen s
an
a ginine
auxo ophic
mu an
om
he
same
o ganism
was
ound
o
include
an
open
eading
ame
encoding
a
427- esidue
polypep ide
ha
is
homologous
o
N-ace ylo ni hine
amino ans e ase
om
Bacillus
sub ilis,
Esche ichia
coli,
and
Saccha omyces
ce e isiae.
The
gene
encoding
N-ace ylo ni hine
amino ans e ase
in
bac e ia
has
been
named
a gD.
The
exp ession
o
Anabaena
sp.
s ain
PCC
7120
a gD,
as
well
as
o
a gC,
was
analyzed
a
he
mRNA
le el.
Bo h
genes
we e
ansc ibed
as
monocis onic
mRNAs,
and
hei
exp ession
was
no
a ec ed
by
exogenously
added
a ginine.
P ime
ex ension
analysis
iden i ied
ansc ip ion
s a
poin s
o
bo h
genes
which
we e
p eceded
by
sequences
simila
o
ha
o
he
E.
coli
RNA
polyme ase
c 70
consensus
p omo e .
A
second
ansc ip ion
s a
poin
o
he
a gD
gene
ha
is
no
p eceded
by
a
c 70
consensus
p omo e
was
de ec ed
in
dini ogen-g own
cul u es.
The
cyanobac e ia
a e
a
pho o ophic
g oup
o
eubac e ia
cha ac e ized
by
hei
abili y
o
ca y
ou
oxygenic
pho osyn-
hesis.
Some
ilamen ous
cyanobac e ia
a e
able
o
ix
molec-
ula
ni ogen
wi hin
specialized
cells
called
he e ocys s
which
di e en ia e
a
semi egula
in e als
along
he
ilamen
unde
condi ions
o
ni ogen
dep i a ion
and
ae obiosis
(22).
This
de elopmen al
p ocess
has
b ough
much
a en ion
o
he
mechanisms
modula ing
gene
exp ession
in
cyanobac e ia.
The
RNA
polyme ase
om
he
ege a i e
cells
o
he
he e ocys -
o ming
cyanobac e ium
Anabaena
sp.
s ain
PCC
7120
has
been
cha ac e ized
and
ound
o
con ain
as
he
p incipal
U
ac o
a
subuni
o
52
kDa
(20).
The
sequence
o
he
sigA
gene
encoding
ha
c
ac o
shows
ha
i s
deduced
polypep ide,
SigA,
esembles
he
p incipal
o
ac o s
om
Esche ichia
coli
and
Bacillus
sub ilis
(2).
The
s ain
PCC
7120
RNA
polyme ase
SigA
is
able
o
ecognize
in
i o
p omo e
sequences
simila
o
hose
ecognized
by
he
E.
coli
RNA
polyme ase
u70
(19,
20).
The
ope a ion
o
his
ype
o
p omo e
in
Anabaena
sp.
s ain
PCC
7120
in
i o
has
ecen ly
been
shown
(7).
The
ansc ip-
ional
s a
poin s
( sp)
o
only
a
ela i ely
small
numbe
o
genes
in
Anabaena
spp.
ha e
been
de e mined.
Some
o
hose
genes
bea
p omo e s
which
show
some
esemblance
o
he
E.
coli
C70
consensus
p omo e ,
bu
o he s
do
no
(19).
In
o de
o
ex end
ou
knowledge
o
he
s uc u e
o
cyanobac e ial
p o-
mo e s,
mo e
genes,
bo h
cons i u i e
and
subjec ed
o
some
kind
o
ansc ip ional
egula ion,
should
be
s udied.
In
his
epo ,
we
p esen
he
analysis
o
exp ession
o
wo
genes
ha
u ned
ou
o
be
cons i u i ely
exp essed
in
ege a i e
cells
o
Anabaena
sp.
s ain
PCC
7120.
These
a e
he
a gC
and
a gD
genes
in ol ed
in
he
biosyn hesis
o
a ginine.
Me hods.
Anabaena
sp.
s ain
PCC
7120
and
i s
de i a i e
a ginine
auxo ophic
mu an s
s ains
CS335
and
CS336
we e
g own
pho oau o ophically
as
desc ibed
p e iously
(9).
Con-
juga ion
o
s ain
CS335
wi h
s ains
o
E.
coli
ca ying
plas-
*
Co esponding
au ho .
Mailing
add ess:
Ins i u o
de
Bioquimica
Vege al
y
Fo osin esis,
Uni e sidad
de
Se illa-C.S.I.C.,
Facul ad
de
Biologia,
Apa ado
1113,
E-41080
Se illa,
Spain.
Phone:
34-5-455.70.
86.
Fax:
34-5-462.01.54.
Elec onic
mail
add ess:
[email p o ec ed].
P esen
add ess:
John
Innes
Ins i u e,
Colney
Lane,
No wich
NR4
7UH,
Uni ed
Kingdom.
mids
wi h
cloned
s ain
PCC
7120
DNA
was
pe o med
by
he
me hod
o
Wolk
e
al.
(23)
as
desc ibed
p e iously
(9).
The
gene al
subcloning
s a egy
used
o
he
a gD
gene
was
as
p e iously
desc ibed
o
he
a gC
gene
(9).
DNA
manipula-
ions
and
cons uc ion
o
plasmids
o
complemen a ion
o
s ain
CS335
and
o
sequencing
we e
ca ied
ou
by
s anda d
p ocedu es
(1,
17).
Sequencing
was
ca ied
ou
by
he
me hod
o
Sange
e
al.
(18)
wi h
exonuclease
III-gene a ed
dele ions
o
wi h
he
aid
o
syn he ic
oligonucleo ides.
The
collec ion
o
E.
coli
a g
mu an s
ha
was
used
o
es
complemen a ion
by
s ain
PCC
7120
DNA
has
been
epo ed
be o e
(9).
To al
DNA
om
s ain
PCC
7120
was
isola ed
as
desc ibed
by
Cai
and
Wolk
(3).
T ans e
o
elec opho e ically
sepa a ed
DNA
agmen s
o
nylon
memb anes
(Hybond-N+
om
Ame sham
o
GeneSc een
Plus
om
DuPon )
and
hyb idiza ion
unde
high-s ingency
condi ions
we e
ca ied
ou
as
ecommended
by
he
memb ane
manu ac u e s.
RNA
was
isola ed
om
exponen ially
g owing
cul u es
o
Anabaena
sp.
s ain
PCC
7120
as
desc ibed
by
Golden
e
al.
(10).
A e
elec opho esis,
ans e
o
RNA
o
nylon
memb anes
(Hybond-N'
om
Ame sham
o
GeneSc een
Plus
om
DuPon )
and
hyb idiza-
ion
we e
ca ied
ou
as
ecommended
by
he
memb ane
manu ac u e s.
P obes
used
in
he
Sou he n
and
No he n
(RNA)
blo
analyses
we e
labelled
wi h
32P
by
using
a
andom
p imed
DNA
labeling
ki
(Boeh inge
Mannheim)
and
[cL-32P]
dCTP.
The
5'
ends
o
he
a gC
and
a gD
ansc ip s
we e
iden i ied
by
p ime
ex ension
(1)
wi h
a ian
myeloblas osis
i us
e e se
ansc ip ase
(Boeh inge
Mannheim).
A
o al
o
50
,g
o
RNA
was
used
in
each
eac ion.
The
oligonucleo ides
used
as
p ime s,
which
we e
labelled
wi h
32P
by
using
polynu-
cleo ide
kinase
and
[,y-32P]dATP,
a e
desc ibed
below.
Iden i ica ion
o
he
Anabaena
sp.
s ain
PCC
7120
a gD
gene.
We
ha e
cloned
p e iously
by
complemen a ion
o
s ain
PCC
7120
a ginine
auxo ophs
wo
genes
in ol ed
in
he
biosyn hesis
o
a ginine
in
Anabaena
sp.
s ain
PCC
7120
(9).
One
o
hose
genes
has
been
iden i ied
as
a gC
encoding
N-ace ylglu ama e
semialdehyde
dehyd ogenase
(9).
The
iden-
i ica ion
o
he
second
gene
cloned
is
p esen ed
he e.
S a ing
om
cosmid
pCSB335-2,
which
ca ies
ca.
31
kb
o
s ain
PCC
7120
DNA
(Fig.
1A)
and
complemen s
he
a ginine
auxo o-
phic
s ain
CS335,
we
subcloned
DNA
agmen s
ha
comple-
6397
6398
NOTES
A
H
S
A
R
Bs
R
X
E
R
I
%'le
I~~~
I
I
I
I I
a gD
a gD
B
H
C
Hp
I
I
l
Hp
P
C
a gC
FIG.
1.
Res ic ion
maps
o
he
genomic
egions
s ain
PCC
7120
con aining
he
a gD
(A)
and
a gC
op
map
o
panel
A,
only
he
wo
HindIII
si es
expanded
egion
a e
shown.
Res ic ion
endonuclea
A,AccI;
B,
BglII;
Bs,
Bs XI;
C,
ClaI;
E,
EcoRI;
H,
H
P,
P uII;
R,
EcoRV;
S,
Spel;
X,
XbaI.
men ed
his
auxo oph.
Fo
subcloning,
agmen s
om
a
pa ial
diges ion
o
pCSB335-2
wi h
Sau3AI
we e
liga ed
o
EB
BamHI-diges ed
posi i e
selec ion
ec o
pRL178
(8).
This
ec o
is
bom
+
and
he e o e
can
be
ans e ed
om
E.
coli
o
3
kb
Anabaena
sp.
s ain
PCC
7120
by
conjuga ion
(23),
hus
pe mi ing
he
es ing
o
complemen a ion
o
he
auxo ophic
ecipien
s ain
by
cloned
DNA
agmen s.
(Howe e ,
pRL178
is
unable
o
eplica e
in
cyanobac e ia;
complemen a ion
H
he e o e
elies
upon
ecombina ion
be ween
he
cloned
DNA
agmen
and
he
cyanobac e ial
genome.)
F om
a
comple-
0.5
kb
men ing
Sau3AI
agmen ,
dele ions
wi h
exonuclease
III
we e
gene a ed,
and
a
agmen
ha
included
mos
o
he
2.4-kb
HindIII-EcoRV
agmen
shown
in
Fig.
1A
and
ha
was
able
o
complemen
s ain
CS335
was
iden i ied.
The
sequence
o
2,259
bp
o
DNA
in
his
egion
o
he
s ain
PCC
7120
genome
was
de e mined.
H
Analysis
o
he
sequenced
segmen
showed
he
p esence
o
U
an
open
eading
ame
(ORF)
o
1,284
nucleo ides
s a ing
0.5
kb
wi h
a
GTG
codon.
A
pu a i e
ibosome
binding
si e
(AG
GAGG)
is
ound
7
nucleo ides
(n )
ups eam
o
he
pu a i e
o
Anabaena
sp.
ansla ion
s a
si e
(Fig.
2A).
Downs eam
o
he
s op
codon,
(B)
genes.
In
he
an
in e ed
epea
ha
is
capable
o
o ming
a
s em-loop
ha
delim
he
s uc u e
and
ha
bea s
se e al
T
(U
in
mRNA)
esidues
in
i s
idIIbIb Hpa
ipai
3'
end
is
ound
(Fig.
2A).
The
ee
ene gy
o
o ma ion
o
his
'd
s uc u e,
which
could
ac
as
a
ho-independen
ansc ip ion
e mina o ,
is
-26.3
kcal/mol
(-110.0
kJ/mol).
The
polypep-
ide
deduced
om
ha
ORF
consis s
o
427
amino
acids
and
A
(5'
egion)
CCATATCTACGGTAGCTAAGGTAATAACTGTCATGTAAGCTGCATCTTCCCATGACCAACCCTCAAACAAAGCGATACCACAAAGTACCGAGAATTAATAC
~sp
(BGli0)
-35
ACCACCGAGAGCGATCGCCCCGGCCATTAACTCTTTTTGGATGCGTTGGTATTTCTGTTCAAGAGTT)AATACAAAATTATTTTCCACTATTGCCRCAGG
-lo
ap
(BGll
and
BG11o)
TTTGAGGATATATAATGTTAAG=CTCAAGAGAAAGTAAGAAACACAATAAATAAAAATTTATTTTTAAGAAACTCTAGGAAACTAGTAAGTAAGTATACAA
RBS
GTG
AGC
CTA
CAA
ACT
CTC
ATT
GAG CAA
WCC
ACG
AAC
CCC
CCA
GAG
TCA
GGT
TCT
GCT
Me
se
leu
gin
h
leu
ile
glu
gin
ala
h
asn
p o
p o
glu
se
gly
se
ala
(3'
egion)
GAA
ATC
AAC
ACC
GCC
TTA
AAA
TTA CTG
GAA
AAA
GCA
TTA
GCA
ACT
GTG ACA
GCG
TAA
ATCATAGACTGCTGGCAAAAATAGCG
glu
ile
asn
h
ala
leu
lys
leu
leu
glu
lys
ala
leu
ala
h
al
h
ala
OCH
B
(5'
egion)
AAGCTTGTGTGTTTTCCATCTGTGAATAAAAATTTTATTTTTTATTGGGAAAATTGTCTGGGATTAGCATTAAGCACTCTATCATTTTTGTCCTATTGCCCT
-35
-10
ap
(BGll
and
BGl1O)
TCGGATAATTTATTTTTTGGTGTTCCCTAAGGTAAATTCAGT&TACAAATGCTGAA&CCATT
ATG
AAT
AAA
CCG
AAA
ATT
TTT
ATT
GAT
RBS
Me
Asn
Lys
P o
Lys
Ile
Phe
Ile
Asp
GGG
GAA
GCG
GGA
ACT
ACA
GGC
TTA
CAG
ATT
TAC
TCA
CGC
CTC
AAC
GAG
CGA
GAT
GAT ATT
GAG
CTA
GTT
AGT
ATT
GCA
Leu
AsnGlu
A g
Asp
Asp
Ile
Glu
Leu
Val
Se
Ile
Ala
Gly
Glu
Ala
Gly
Th
Th
Gly
Leu
Gln
Ile
Ty
Se
A g
FIG.
2.
Nucleo ide
sequences
o
he
5'
ends
o
he
a gD
(A)
and
a gC
(B)
genes
o
Anabaena
sp.
s ain
PCC
7120,
he
egions
ups eam
o
hese
genes,
he
3'
end
o
he
a gD
gene,
and
he
egion
downs eam
o
his
gene
(A).
Some
pu a i e
ansc ip ion
s a
poin s
( sp)
ound
wi h
RNA
isola ed
om
cul u es
g own
on
BG11
and/o
BG
10
medium
a e
indica ed
(bold ace
and
unde lined).
Pu a i e
-35
and
-10
p omo e
sequences
ound
ups eam
om
he
sps
used
in
bo h
BG11
and
BG110
medium
a e
indica ed
in
bold ace.
Pu a i e
ibosome
binding
si es
(RBS)
a e
unde lined.
Wi hin
he
coding
sequence
o
he genes,
he
segmen s
complemen a y
o
he
oligonucleo ides
used
o
p ime
ex ension
analysis
a e
unde lined.
In
he
egion
downs eam
o
he
3'
end
o
he
a gD
gene,
sequences
ha
may
be
in ol ed
in
he
o ma ion
o
a
s em-loop
s uc u e
a e
unde lined.
AAAAAATATCCCAAATGTAGGGTGCGTCAGTGTGATATACCCTAACTATAGGCAGAAATTATTCATGCTGACGCAACCTACCCAACTACCAAACCATCGATT
J.
BA=-1RIOL.
NOTES
6399
has
a
p edic ed
molecula
weigh
o
46,110.
A
sea ch
o
p o eins
wi h
simila
sequences
in
he
a ailable
da abases
e ealed
ha
he
deduced
polypep ide
had
homology
o
nu-
me ous
amino ans e ases
om
e y
di e se
biological
sou ces.
The
p o eins
mos
simila o
he
s ain
PCC
7120
polypep ide
we e
he
N-ace ylo ni hine
amino ans e ase
en-
zymes
om
B.
sub ilis
(16),
Saccha omyces
ce e isiae
(12),
and
E.
coli
(12).
These
p o eins
a e
o
a
size
simila
o
ha
o
he
deduced
s ain
PCC
7120
polypep ide
and
show
iden i ies
o
44%
(B.
sub ilis),
41%
(S.
ce e isiae),
and
41%
(E.
coli)
wi h
ha
polypep ide.
The
gene
encoding
N-ace ylo ni hine
amino ans e -
ase
(EC
2.6.1.11)
in
bac e ia
has
been
named
a gD.
The
A gD
p o ein
ca alyzes
he
ans o ma ion
o
N-ace ylglu ama e
semialdehyde
in o
N-ace ylo ni hine
by
means
o
a
ansami-
na ion.
This
is
he
ou h
s ep
in
he
pa hway
o
biosyn hesis
o
a ginine
om
glu ama e
(4).
The
iden i ica ion
o
he
se-
quenced
gene
om
Anabaena
sp.
s ain
PCC
7120
as
a gD
is
consis en
wi h
he
pheno ype
o
mu an
s ain
CS335,
which
was
able
o
g ow
on
o ni hine
(which
is
syn hesized
om
N-ace ylo ni hine
[4])
bu
no
on
glu ama e
(9).
Amino ans-
e ases
ca alyze
he
e e sible
ans e
o
amino
g oups
om
amino
acids
o
oxo
acids
by
using
py idoxal
phospha e
as
a
co ac o .
Py idoxal
phospha e
in e ac s
wi h
he
£-amino
g oup
o
a
Lys
esidue
o
o m
a
Schi
base.
A
sea ch
o
unc ional
domains
in
s ain
PCC
7120
A gD
by
using
he
p og am
MOTIFS
o
he
Gene ics
Compu e
G oup
package
(6)
iden-
i ied
a
pu a i e
py idoxal
phospha e
binding
si e
in
which
Lys-277
would
be
in ol ed
in
Schi
base
o ma ion.
The
a gC
and
a gD
genes
a e
no
clus e ed
in
he
genome
o
Anabaena
sp.
s ain
PCC
7120.
Sou he n
blo
analysis
o
o al
DNA
isola ed
om
s ain
PCC
7120
diges ed
wi h
HindIII
showed
a
single
s ong
hyb idiza ion
band
o
abou
5
kb
when
he
HindIII
agmen
ca ying
he
s ain
PCC
7120
a gD
gene
(Fig.
1A)
was
used
as
a
p obe
and
a
single
hyb idiza ion
band
o
2.6
kb
when
a
p obe
o
he
a gC
gene
( he
1.6-kb
ClaI
agmen
[Fig.
1B])
was
used
(da a
no
shown).
An
addi ional,
sligh ly
hyb idizing
band
o
2.6
kb
ha
migh
co espond
o
sequences
encoding
ano he
amino ans e ase
was
obse ed
wi h
he
a gD
p obe.
I
appea s,
he e o e,
ha
a gC
and
a gD
a e
single-copy
genes
in
Anabaena
sp.
s ain
PCC
7120,
which
is
consis en
wi h
he
ac
ha
s ain
PCC
7120
mu an s
al e ed
in
hose
genes
ha e
been
eadily
isola ed
(9).
No
e idence
o
clus e ing
o
he
a gC
and
a gD
genes
in
he
s ain
PCC
7120
genome
has
been
ound.
Thus
(i)
none
o he
cosmids
ca ying
s ain
PCC
7120
DNA
ha
we e
able
o
complemen
he
a gC
mu an
(s ain
CS336)
was
able
o
complemen
he
a gD
mu an
(s ain
CS335)
and,
con e sely,
he
CS335-complemen ing
cosmids
we e
unable
o
comple-
men
s ain
CS336
(9);
and
(ii)
no
c oss-hyb idiza ion
be ween
pCSB335-2
and
CS336-complemen ing
cosmids
was
obse ed
(no
shown).
This
da a
oge he
wi h
a
compa ison
o
he
es ic ion
map
o
he
inse
o
cosmid
pCSB335-2
(Fig.
1A)
wi h
hose
o
he
inse s
o
cosmids
isola ed
om
comple-
men ed
exconjugan s
o
he
a gC
mu an
(s ain
CS336)
(9)
sugges
ha
he
HindIII
agmen s
con aining
he
a gC
and
a gD
genes
a e
a
leas
9
kb
apa
in
he
genome
o
s ain
PCC
7120.
Wi h
ega d
o
o he
a ginine
biosyn hesis
(a g)
genes,
he
DNA
sequences
lanking
a gD
ha
we e
de e mined
(ca.
400
bp
5'
o
he
gene
and
ca.
500
bp
3'
o
he
gene)
do
no
seem
o
encode
any
p o ein
ela ed
o
a ginine
biosyn hesis,
and
an
ORF
ha
is
ound
60
bp
downs eam
o
a gC
(accession
numbe
X65511
in
he
EMBI'GenBank/DDBJ
nucleo ide
sequence
da a
lib a ies)
would
code
o
a
p oduc
un ela ed
o
a ginine
me abolism.
(This
ORF,
o
which
only
178
codons
we e
sequenced,
has
56%
iden i y
in
an
o e lap
o
174
amino
A
1.5
kb-p
B
1.1
kb-
+
--M
-
-
<
^:
A
T~
Lwo
H,21
i
AS
,
Q
>P2-w
M
-7.4
-5.3
-2.8
-1.9
-1.6
-1.0
-0.6
-0.4
-0.3
-1.9
-1.6
1.0
-0.6
0.4
FIG.
3.
No he n
blo
analysis
o
he
exp ession
o
he
a gD
(A)
and
a gC
(B)
genes
in
Anabaena
sp.
s ain
PCC
7120.
To al
RNA
isola ed
om
cells
g own
in
medium
BG11
supplemen ed
o
no
wi h
5
mM
L-a ginine
was
used
(25
pug
in
each
lane).
The
posi ion
and
sizes
(in
kilobases)
o
some
RNA
molecula
weigh
s anda ds
a e
indica ed.
acids
wi h
o389
o
E.
coli,
an
ORF
which
is
loca ed
be ween
wo
genes
in ol ed
in
he
biosyn hesis
o
en e obac e ial
common
an igen
[5].)
Addi ionally,
in
complemen a ion
assays
o
a
collec ion
o
s ains
o
E.
coli
mu a ed
in
di e en
a ginine
biosyn hesis
genes,
we
did
no
obse e
complemen a ion
o
any
E.
coli
mu an
by
he
a gD-ca ying
cosmid
pCSB335-2,
whe eas
he
a gC-ca ying
cosmid
pCSB336-24
(9)
was
able
o
complemen
only
an
E.
coli
a gC
mu an
(da a
no
shown).
(In
his
s udy,
no
a gD
mu an
o
E.
coli
was
es ed
because
E.
coli
a gD
mu an s
a e
leaky.)
These
esul s
sugges
ha
in
he
genome
o
Anabaena
sp.
s ain
PCC
7120,
nei he
a gC
no
a gD
is
lanked
by
o he
a g
genes.
Analysis
o
a gC
and
a gD
mRNAs.
RNA
was
isola ed
om
cul u es
o
Anabaena
sp.
s ain
PCC
7120
g own
on
BG11
medium
supplemen ed
o
no
wi h
5
mM
L-a ginine,
which
is
an
amino
acid
ha
is
eadily
aken
up
by
cells
o
s ain
PCC
7120
(13).
No he n
blo
analysis
was
pe o med
by
using
as
a
p obe
an
EcoRV
agmen
o
0.9
kb
ha
con ains
he
3'
egion
o
a gD
(Fig.
1A)
o
an
HpaI
agmen
o
0.6
kb
in e nal
o
he
a gC
gene
(Fig.
1B).
Single
hyb idizing
RNAs
o
abou
1.5
kb
(Fig.
3A)
and
1.1
kb
(Fig.
3B)
we e
obse ed
wi h
he
a gD
and
a gC
p obes,
espec i ely.
Because
he
a gD
and
a gC
genes
consis
o
1,284
and
966
bp,
espec i ely,
bo h
genes
appea
o
be
ansc ibed
as
monocis onic
mRNAs.
No
di e ence
be-
ween
he
RNA
p epa a ions
isola ed
om
cul u es
supple-
men ed
wi h
a ginine
and
hose
om
cul u es
no
supple-
VOL.
176,
1994
6400
NOTES
men ed
wi h
a ginine
was
obse ed.
In
he
case
o
he
a gC
mRNA,
he
lack
o
e ec
o
exogenously
added
5
mM
L-
a ginine
was
co obo a ed
by
p ime
ex ension
analysis
(da a
no
shown).
The
lack
o
ep ession
o
he
a gC
and
a gD
genes
by
a ginine
is
consis en
wi h
epo ed
da a
on
he
ac i i ies
o
a ginine
biosyn hesis
enzymes
inAnabaena
a iabilis
and
o he
cyanobac e ia
(15).
The e o e,
in
he
cyanobac e ia,
he
only
egula o y
mechanism
ound
o
da e
o
ope a e
o
a ginine
biosyn hesis
is
eedback
inhibi ion
o
N-ace ylglu ama e
phos-
pho ans e ase
(14),
a
egula o y
mechanism
ha
is
ypical
in
o ganisms
ha ,
like
he
cyanobac e ia,
exhibi
he
cyclic
pa h-
way
o
o ni hine
syn hesis
(4).
P omo e
s uc u e.
The
5'
endpoin s
o
mRNAs
o
he
a gC
and
a gD
genes
in
Anabaena
sp.
s ain
PCC
7120
we e
de e -
mined
by
p ime
ex ension
analysis.
The
p ime s
used
a e
depic ed
in
Fig.
2A
and
B
o
a gD
and
a gC,
espec i ely.
An
ex ension
p oduc
ha
can
ep esen
a
ansc ip ion
s a
poin
( sp)
was
ound
103
bp
ups eam
om
he
s a
o
he
a gD
gene
when
RNA
isola ed
om
cul u es
g own
in
BG11
(in
he
p esence
o
ni a e)
o
BG110
(wi h
no
sou ce
o
combined
ni ogen)
was
used
(Fig.
4A;
see
also
Fig.
2A).
Wi h
RNA
isola ed
om
BG110-g own
cul u es,
an
addi ional,
la ge
ex ension
p oduc
was
obse ed
(Fig.
4A;
see
also
Fig.
2A).
This
ex ension
p oduc
could
ep esen
a
sp
loca ed
158
bp
ups eam
om
he
s a
o
he
a gD
gene,
hough
he
possibil-
i y
ha
i
o igina ed
om
deg ada ion
o
a
la ge
RNA
canno
be
uled
ou .
Wi h
ega d
o
a gC,
an
ex ension
p oduc
ha
can
ep esen
a
sp
was
ound
20
bp
ups eam
om
he
s a
o
he
gene
(Fig.
4B;
see
also
Fig.
2B).
Simila
esul s
o
p ime
ex ension
assays
o
a gC
we e
ob ained
wi h
RNA
isola ed
om
cul u es
g own
in
ei he
BG11
o
BG110
medium
(Fig.
4B).
Se e al
5'
endpoin s
co esponding
o
smalle
RNAs
a e
no iceable
o
he
eac ions
shown
in
Fig.
4A
and
B.
They
could
be
due
o
pa ial
RNA
deg ada ion
o
incomple e
ex ension
o
he
p ime s.
Ups eam
om
he
pu a i e
a gC
sp
and
om
he
pu a i e
main
sp
o
a gD,
sequences
ha
esemble
he
consensus
sequence
o
p omo e s
ecognized
by
he
E.
coli
RNA
poly-
me ase
u70
(TTGACA[16
o
18
n ]TATAAT[5
o
9
n ]- sp)
a e
ound.
These
sequences
ead
as
ollows:
'lT
lATT(18
n )TA
AAAT(5
n )- sp
(a gC)
and
TTGCCA(16
n )TAATGT(4
n )-
sp
(a gD)
(Fig.
2).
(O e lapping
wi h
he
pu a i e
-10
box
o
he
a gD
p omo e ,
he
sequence
TATAAT,
which
ep esen s
a
pe ec
P ibnow
box,
is
ound;
howe e ,
his
hexame
is
sepa a ed
by
only
14
n
om
he
pu a i e
-35
box
o
he
p omo e .)
The e o e,
hese
a gC
and
a gD
p omo e s,
which
a e
cons i u i e,
may
be
ecognized
by
he
Anabaena
sp.
s ain
PCC
7120
RNA
polyme ase
ca ying
he
p incipal
a
ac o ,
SigA,
which
has
been
shown
o
ecognize
p omo e s
simila
o
he
E.
coli
RNA
polyme ase
u 70
consensus
p omo e
(19).
I
is
in e es ing
ha
an
RNA
species
mo e
abundan
in
dini ogen- ixing
(BG110
medium)
han
in
ni a e-g own
(BG11
medium)
cul u es
o
Anabaena
sp.
s ain
PCC
7120
was
de ec ed
wi h
he
a gD
p ime
(Fig.
4A).
We
canno
exclude
ha
a
BG110-speci ic
RNA
can
also
exis
o
a gC,
since
he
a gC
mRNA
was
di icul
o
de ec
and
a
low-abundance
ex ension
p oduc
could
ha e
been
unno iced.
In
s ain
PCC
7120,
some
genes,
e.g.,
sigA
o
glnA,
ha e
been
shown
o
be
ansc ibed
om
se e al
p omo e s
(2,
21).
I
is
possible
ha
genes
ha
a e
exp essed
in
bo h
he e ocys s
and
ege a i e
cells
a e
ansc ibed
om
di e en
p omo e s
in
he
wo
cell
ypes.
Al hough
a
numbe
o
genes
ha
a e
exp essed
speci -
ically
in
he e ocys s
ha e
been
in es iga ed
(e.g.,
ni ogen
ixa ion
genes),
a
consensus
sequence
o
p omo e s
unc ional
in
he e ocys s
has
no
ye
been
de i ed.
A ginine
biosyn hesis
laiT
C
T
T
C
T
C
A
A
4-
C
T
T
A
T
G
C
A
A
T
T
C
A
G
T
T
C
T
'c
A
-
-
T
C
0
A
T
T
A
A
G
T
C
A
s..
G
4-,
B
FIG.
4.
Iden i ica ion
o
he
5'
ends
o
heAnabaena
sp.
s ain
PCC
7120
a gD
(A)
and
a gC
(B)
ansc ip s.
RNA
p epa ed
om
cells
g own
on
BG11
o
BG110
medium
was
used
in
p ime
ex ension
expe imen s.
The
p ime -ex ended
p oduc s
we e
coelec opho esed
wi h
a
sequencing
ladde
gene a ed
wi h
he
same
oligonucleo ide
p ime .
The
a ows
on
he
igh
o
each
panel
indica e
he
posi ions
o
pu a i e
sps.
The
nucleo ide
sequences
a ound
hese
pu a i e
sps
a e
also
p esen ed
(no e
ha
he
sequence
o
he
s ands
complemen a y
o
hose
p esen ed
in
Fig.
2
a e
shown).
enzymes,
including
N-ace ylo ni hine
amino ans e ase
(A gD),
a e
p esen
in
Anabaena
spp.
in
he e ocys s
as
well
as
in
ege a i e
cells
(11).
We
do
no
know,
howe e ,
whe he
he
uppe
band
ound
wi h
RNA
p epa a ions
om
cul u es
g own
in
BG110
medium
co esponds
o
he e ocys -localized
mRNA
molecules.
This
RNA
species
was
also
de ec able,
al hough
in
educed
amoun s,
wi h
RNA
p epa a ions
om
BG11-g own
cul u es,
bu
i
should
be
no ed
ha
BG11-g own
cul u es
o
J.
BAc- ERIOL.
VOL.
176,
1994
Anabaena
sp.
s ain
PCC
7120
a e
no
comple ely
de oid
o
he e ocys s.
Nucleo ide
sequence
accession
numbe .
The
sequence
e-
po ed
in
his
no e
o
2,259
bp
o
DNA
o
he
s ain
PCC
7120
genome
will
appea
in
he
EMBL/GenBank/DDBJ
da a
lib a -
ies
unde
accession
numbe
X78854.
This
wo k
was
suppo ed
by
g an
PB90-0114
om
Di ecci6n
Gene al
de
In es igaci6n
Cien i ica
y
Tecnica,
Spain.
B.F.
was
he
ecipien
o
a
ellowship
om
Plan
de
Fo maci6n
de
Pe sonal
In es-
igado
(Minis e io
de
Educaci6n
y
Ciencia),
Spain.
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NOTES
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