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DNA RESEARCH 5, 121-126 (1998)
Short Communication
An 8 kb Nucleotide Sequence at the 3' Flanking Region of the
sspC Gene (184°) on the Bacillus subtilis 168 Chromosome
Containing an Intein and an Intron
Bacterial Molecular Genetics RU, Korea Research Institute of Biosaence and Biotechnology (KRIBB),
P.O. Box 115, Yusong, Taejon 305-600, Korea
(Received 2 March 1998)
Abstract
As part of the Bacillus subtilis genome sequencing project, we determined the complete nucleotide
sequence of an 8000-bp fragment downstream of the sspC gene (184°) of the B. subtilis 168 chromosome.
The sequence analysis shows that the sspC gene is located inside of the SP(3 region, which differs from
the current genetic map of B. subtilis 168. This region contains 12 putative ORFs (yojQ through yojZ
and sspC). A homology search for the deduced products of the ORFs shows significant similarities to
enzymes involved in deoxyribonucleotide metabolism: ribonucleotide reductase (Nrd) E, NrdF, thioredoxin
and dUTPase. Interestingly, this DNA fragment includes two split genes, yojP containing conserved motifs
of an intein and yojQ and yojS with an 808-bp intervening sequence for a putative intron structure. In
addition, the yojR gene includes a putative new DNA replication terminator.
Key words: Bacillus subtilis; genome sequencing; yojP-sspC region; intein; intron.
Inteins and introns as splicing elements have
been well characterized in eukaryotic organelles and
bacteriophages.1'2 It had been once considered that inteins and introns were unique to eukaryotes. But many
forms of intervening genes have also been identified in
archea and bacteria. Bacterial inteins have been found
in the dnaB gene of Synechocystis and in the recA and
gyrA genes of Mycobacterium species.3 Prokaryotic group
I introns have been extensively studied in the virulent
bacteriophage T4. Two Bacillus introns have been reported in bacteriophages SPO1 and /322, respectively.4'5
Both belong to the self-splicing group I introns and contain an open reading frame (ORF) that encodes a homing
endonuclease.6
We cloned a DNA fragment at the 3' flanking region
of the sspC gene at 184° of the B. subtilis chromosome.7
Here we report the sequencing results of the 8-kb region
containing the sspClocus. Sequence analysis showed that
the fragment includes two split genes encoding homologues of the large and small subunits of ribonucleotide
reductase with intervening sequences. Each of the intervening sequences displays characteristic features of inteins in its amino acid sequence and introns both in its
*
f
Communicated by Naotake Ogasawara
To whom correspondence should be addressed. Tel. +8242-860-4412, Fax. +82-42-860-4594, E-mail: shpark@kribb
4680.kribb.re.kr
The GenBank accession number for the nucleotide sequence
reported in this paper is AF012906.
primary sequence and in its potential for forming secondary structure, respectively. Even though the complete sequence of B. subtilis 168 genome was reported,8
those intervening sequences have not been described.
1.
Cloning of the 3' Flanking Region of sspC
We obtained two plasmids: pSQ5 containing sspC
(184°) of the B. subtilis chromosome and pSQ52 containing an 8-kb fragment at the 3' flanking region of
the sspC gene (Fig. 1). On the basis of the published
DNA sequence of the B. subtilis sspC gene, polymerase
chain reaction (PCR) primers (Cl-F and Cl-R; shown in
Fig. 1) were synthesized and used to amplify a 730-bp
PCR product using the B. subtilis chromosomal DNA as
a template. The PCR product was cloned into the vector pDIA5304, yielding pSQ5. Chromosomal walking to
obtain plasmid pSQ52 from pSQ5 was performed by the
plasmid rescue method.28
2.
Nucleotide Sequencing and Identification of
ORFs
The complete nucleotide sequence of an 8000-bp DNA
fragment containing the sspC gene was determined; its
physical organization is shown in Fig. 1. Our 8-kb
sequence deposited under the GenBank accession no.
AF012906 is identical to the corresponding part of the en-
Downloaded from http://dnaresearch.oxfordjournals.org/ at Korea Research Institute of Bioscience & Biotechnology on January 13, 2012
Sa-Youl GHIM, Soo-Keun CHOI, Byung-Sik SHIN, and Seung-Hwan PARK*
The 184° Region of the B. subtilis Chromosome.
122
[Vol. 5,
184°
185°
8 kb
pSQ52
pSQ5 ^ ^ H
\G - -1 5 k c a i m o H
K
E
S A
Pv
N
I
I I
I
l__l
Primer
ND-F
yojQ
yojR
P
B N B " E
LJJ
I
"
\G - -12 1 Kcal moi-1
Ni
Ni
BgE"
I
I
U
Bg
1
yojT
yojV
yoj X
yojZ
yojS yojU
yojW yojY
sspC
Figure 1. Physical map and gene organization of the B. subtilis genomic region containing the sspC gene (184°).7 The vector used was
pDIA530428 for both pSQ5 and pSQ52 and the location of the cloned DNA in pDIA5304 is indicated with solid bars. Restriction
sites are abbreviated as K, Kpn I; E, EcoRl; P, Pst I; S, Sal I; A, Aat II; Pv, Pvu I; N, Nsp I; B, Bst EII; Ni, Nsi I; and Bg, Bgl II.
Three stem-loops indicate putative attenuator or terminator structures with their free energy.29 The letters below each thick arrows
and a solid bar represent the name of the ORF and the arrowheads shows the direction of transcription and translation. Primers for
PCR are indicated by arrows and letters right below the solid bar.
Table 1. Similarity of the predicted ORF Products to other known proteins.
Gene nami
GenBank
SubtiUst
Endpoints
Database
(nucleotides)
accession no
Description or similar protein in database
bl
BLAST
Percentage
score
identity0)
yo/P
yosO
1>1986
SP P50621
Ribonucleotide reductase E (NrdE) of B. subtilis
1325
77(319)
yoiQ
yosP
2316>2896
SP. P50621
N-termmal region of ribonucleotide reductase F
(NrdF) of B. subtilis
955
97(193)
yoiR
yosO
3008>3529
E X67865
PIR S50094
PIR S50092
Orf36 1 of bacteriophage SPP1
Endodeoxyribonuclease of bacteriophage SP82
Endodeoxyribonuclease of bacteriophage phi-E
124
102
97
23(173)
23(173)
24(173)
3705M113
SP P50621
C-terminal region of ribonucleotide reductase F
(NrdF) of B subtilis
473
77(136)
4110>4352
E: X92690
Glutaredoxin-like protein (NrdH) of Lactococcus
lactis
Thioredoxin (TrxA) of B subtilis
Thioredoxin (TrxA) of Alicyclobacillus
acidocaldarius
Thioredoxin (TrxA) of Streptomyces clavuligerus
74
25 (80)
54
58
39(80)
29(80)
yojS
yojT
yosR
SP P14949
SP P80579
SP Q05739
yojU
yosS
4398>4826
GB U38906
E Z96072
yosT
yojV
yojW
yo/X
yoiY
yojZ
yosV
yosW
yosX
4921>5370
5807>5974
5975>6265
6418>6753
6984>7337
yokA
sspC
7855<7637
dUTPase of the temperate lactococcal
bacteriophage r1t
Dut of Mycobactenum tuberculosis
56
30(80)
117
32(142)
115
32(142)
301
100 (72)
None
None
None
None
None
SP P02958
Small, acid-soluble spore protein C (SspC) of
B. subtilis
a) Our data in GenBank; data in SubtiList of Institut Pasteur.
b) SP, SwissProt; E, EMBL; PIR, NBRF-PIR; GB, GenBank.
c) The number of amino acids over which the percentage match was determined is shown in parentheses.
tire nucleotide sequence of B. subtilis genome.8 Putative ORFs; with the exception of yojP and yojS, all met the
ORFs initiating with ATG, GTG, or TTG codons pre- above criteria. Three putative attenuator or terminator
ceded by a typical ribosome binding site were searched for structures were identified within the region (depicted in
in all six translation frames.9 As summarized in Table 1, Fig. 1 as stem-loop structures),
analysis of the sequenced region revealed 12 putative
Our sequence analysis indicates that the sspC gene is
Downloaded from http://dnaresearch.oxfordjournals.org/ at Korea Research Institute of Bioscience & Biotechnology on January 13, 2012
yojP
P
\G - - i s 3 Heal moH
No. 2]
Similarity of the Putative ORF Products to
Known Proteins
Results of homology searches for deduced products of
ORFs are summarized in Table 1. Among 12 ORFs, sspC
was the only known B. subtilis gene mapped and characterized in this region.7 The putative products of yojV,
yojW, yojX, yojYand yojZshowed no significant homology to any known proteins in the data banks. General
features of the five putative gene products that showed
homology to other known proteins are discussed below.
The putative product of the partial ORF of yojP shows
significant homology to the product of the large subunit
of ribonucleotide reductase (NrdE) located at 168° on the
chromosome of B. subtilis.12 These proteins share 77%
identity in a 319-amino acid (aa) overlap. Table 2 indicates that the yojP product is more similar to NrdE than
to NrdA of the class I ribonucleotide reductases, which
are known to be essential enzymes for the biosynthesis of
deoxyribonucleotides in bacteria.13'14
The putative product of yojQ shows striking homology
to the N-terminal region of the small subunit of ribonucleotide reductase (NrdF) of B. subtilis.12 These products
share 97% identity in a 193-aa overlap. Careful examination of the nucleotide sequence data revealed an unusual
putative ORF, yojS, which lacks a recognizable ribosome
binding site. The deduced amino acid sequence of yojS
also showed strong homology with the C-terminal region
of B. subtilis NrdF, having 77% identity in a 136-aa overlap. The amino acid sequence of the proposed ORF
from yojQ/S, without the intervening region, matched
perfectly with that of B. subtilis NrdF. The amino acid
identity between the putative YojQ/YojS and NrdF of
B. subtilis is 86%. Table 2 indicates that the yojQ/S
product is more similar to NrdF than to NrdB of the
class I ribonucleotide reductases. It is known that the
nrdE/nrdF genes at 168° in B. subtilis specify the large
and small subunits of ribonucleotide reductase, respectively, and that their function is essential,12 unlike the
case in Enterobacteriaceae, from which these genes can
be deleted.15
The two coding sequences of yojQ and yojS were separated by an unexpected intervening region of 808 nucleotides. This intervening region included an ORF
with 173 aa, designated yojR. The yojR gene encodes a 20.1-kDa hypothetical protein which shows
Table 2. Amino acid sequence homology of the large and small
subunits of the class I ribonucleotide reductase (RR) from different sources with YojP and YojQ/S of B. subtilis 168.
% identity of RR subunit with
YojP
YO|Q/S
Reference
or
database accession no a '
8 subtilis NrdE. NrdF
77
86
12
E coli NrdA, NrdB
23
21
30
E coll NrdE, NrdF
45
41
GB: AE000352
Source
a) GB: GenBank.
Consensus for
DNA terminator
Putative terminator
sequence
C G
N
A G
CTCAG T C j A A C
Q^^^G
I
II I II I III I II II II I II I I
TTTGCGACTA ACCTATGTAC TAAATAGTTC
Figure 2. DNA sequence alignment of a putative DNA replication
terminator with the known consensus of B. subtilis DNA terminator. The numbering indicates position of nucleotide residue.
N in the consensus sequence represents any nucleotide. Vertical
lines indicate identical nucleotide residues.
weak homology to some bacteriophage intron-encoded
endodeoxyribonucleases.16 In addition, a new putative
DNA replication terminator was identified in the middle
of the yojR gene (Fig. 2).17 Rowe et al.18 have reported
that DNA cloned from near the replication terminus of
B. subtilis showed strong homology to the DNA of SP/3
prophage.
The yojT gene product shows meaningful homology
to a glutaredoxin-like protein, NrdH, and thioredoxins
(TrxA) from various sources (E. Pedone, unpublished;
SwissPro accession no. P80579),19'20'21 suggesting that it
may function as an electron transporter in ribonucleotide
reduction like NrdH of Lactococcus lactis.19
The putative product of yojU shows significant homology to dUTPases of the temperate lactococcal bacteriophage rlt and Mycobacterium tuberculosis (S. Gentles
and C. M. Churcher, unpublished; EMBL accession no.
Z96072),22 i.e. 32% identity in 142 aa. The dUTPase is
involved in the biosynthesis of dTTP by catalyzing the
conversion of dUTP to dUMP.14
It is very interesting that the genes clustered in the
temperate SP/3 prophage region of the B. subtilis chromosome show significant homology to NrdE, NrdF, an
electron transport protein, and to a dUTPase. These enzymes may be involved in deoxyribonucleotide synthesis
when the SPfl prophage is induced to replicate under
proper conditions.
Downloaded from http://dnaresearch.oxfordjournals.org/ at Korea Research Institute of Bioscience & Biotechnology on January 13, 2012
located within the SP(3 region (S.-Y. Ghim and others,
unpublished; GenBank accession no. AF006665), which
is different from the recent genetic map of B. subtilis.10
The left attachment site for the SP(3 prophage (attSP/3)
has been identified at around 194° of the chromosome.11
Actually, the right attachment site for the SP0 prophage
was found in the C-terminal region of orfE5, which is located around 4 kb upstream of the sspC locus. Thus this
8-kb segment is included in the SP/3 prophage region.
3.
123
S.-Y. Ghim et al.
The 184° Region of the B. subtilis Chromosome.
124
Block B
Consensus
for intein motifs
Putative intein
sequence
G
h hT
Block C
H hhh
GYEIRATEWHKFYV
«
LhG
hhaG
IMGIIAGDG
88
Block D
K IP h
Block E
L GhFahDG
KTRVPEFI
i?6
[Vol. 5,
Block H
Block F
p S hh h LL hGI rVYDLpVa
YLSGLFQTDG
186
SIHYESLQDVQKLLLNMGV
221
Block G
Fh
NGhhhHNp
DVYDTTQEDYHSLI
324
NGIVTGNC
333
4.
Two Proposed Intervening Sequences: Intein
and Intron
The deduced amino acid sequence of the putative ORF
of yojP showed strong homology in its C-terminal region
with that of B. subtilis NrdE but no homology in its Nterminal region where we could find conserved intein motifs. Eight conserved motifs termed Block A-H have been
known in intein sequences.3'23 Seven (Block B-H) out of
eight motifs were found in the deduced amino acid sequence of yojP (Fig. 3). Sequence for Block A motif could
be found in the neighboring amino acid sequence of YosO
in the SubtiList database.8 Highly conserved residues of
one His in Block B, two Gly in Block C and one Asn in
Block G found in all inteins are present in this putative
intein sequence. At the C-terminal splice junction of the
putative intein, a Gly residue found in Ceu clpP and Mja
Rpol A' inteins24'25 was observed instead of a conserved
His of most known inteins.
In the intervening region between yojQ and yojS we
found highly conserved consensus elements called P, Q,
R and S sequences that are generally found in group I
introns (Fig. 4).26 Moreover, the intervening sequence
had the potential of forming a secondary structure similar to that of eukaryotic group I introns (Fig. 5). It
would be interesting we attempted to confirm that the
B. subtilis 168 chromosome actually contains an intervening sequence between the yojQ and yojS regions. The
PCR product amplified using primers ND-F and ND-R
(shown in Fig. 1) and the B. subtilis chromosomal DNA
as a template proved conclusively that an intervening sequence of 808 nucleotides was located between yojQ and
yojS (data not shown). The intervening region includes
an ORF, yojR, of 519 nucleotides in length. Although
the putative product of the yojR gene shows weak homology to endodeoxyribonucleases of various introns, it has
a definite His-X-Asn-X-His protein sequence motif. This
motif has been found in some group I intron-encoded
endonucleases and within the Zn-finger-like domains of
several group II intron ORFs.26'27
Even though we do not yet have direct experimental
P Sequence
Q Sequence
Group I intron
Group IA intron
AUGCUGGAAA
U
A
AAUCAGCAGG
U
C
IMlllllll
MINIMI!
yojR
UUGCUGGAAA
AAUCAGCAGC
R Sequence
S Sequence
Group I intron
Group IA intron
UCAGAGACUAXA
AC
AAGAUAUAGUCC
U
IIIIIMlll
1 111 m m 1
yojR
UCAACGACUAUC
AUGAUAUAGUCU
Consensus:
Figure 4. DNA sequence alignment of the yojR region with highly
conserved P, Q, R and S sequences of eukaryotic group I and
group IA introns. Arrows above sequences indicate regions of
dyad symmetries. Vertical lines indicate identical nucleotide
residues.
evidence for the intein and intron in the intervening regions among yojP, yojQ and yojS, we propose that the
putative intein of yojP region can be posttranslationally
excised to form the complete NrdE homologue, and the
potential secondary structures of yojR region may participate in self-splicing of the mRNA, producing a transcript
encoding the complete NrdF homologue. More detailed
biochemical and physiological studies are need to confirm
the presence of the intein and intron.
Acknowledgements: We gratefully thank Jan
Neuhard, Jens Lykke Andersen and Robert L. Switzer
for critically reading the manuscript, and David A. Shub
and Shmuel Pietrokovski for valuable discussions. This
study was financially supported by the Korean Ministry
of Science and Technology.
Downloaded from http://dnaresearch.oxfordjournals.org/ at Korea Research Institute of Bioscience & Biotechnology on January 13, 2012
Figure 3. Araino acid sequence alignment between the putative product of yojP and conserved intein motifs. The numbering indicates
position of amino acid residue. Vertical lines indicate identical residues. Capital letters in the consensus line indicate conserved
amino acids (standard single letter code); h, hydrophobic residue (G, A, V, L, I or M); a, acidic residue (D or E); p, polar residue
(S, T or C); and r, aromatic residue (F, Y or W).
No. 2]
S.-Y. Ghim et al.
P5
U
u
U-A
^
5
1
A U
U
U
U-A
C-G
C-G
U-A
A
C
IA
A
l
Al
V
//
Gllf
P4 1
C A
G
A
G-C
p 2 c-G
SIA
U-A
U G
G-C
U G
GIC L .AAG
P6a /
U-A
A-U
CUG-CCCUUU-ACGGCAAAUGUGAACCCUCUCUAAUU-AGCG
CUAUGCGA
,3 ""I
I I I
.CGUGGGUGAGAA
/
AA
A
C
P8
iI U
_ _ ^ —
II
I
U-GC
c£-~-^P 7 _ _R_ / A/ c
17 n t
A*
U*
G*
II
GAUACGCU
AA/
P6
1
V
X
488
nt
J
— AUCAGCA
UUCCCCA -—
1
I II
AACGGGAGGCAUCC/— UACAGGGAUGAUGA UAUAGUC-UGCUC-G C G G - C G A-U G A A C U
A
U-A
A-U
C-G
GA
/
A
C-G
U-A
U-A
/
G
C-G
G-C
U-A
GGU-A G C U U A A A-U 1
G
G-C
G-C
C-G
A-U
U G __,
G
A-U
A
G
C-G
A-u P7.2
C-G P7.1
U
A
C
G-C
U-A
G-C
A
G
A
A
C A
A
C-G
G
A
A
G
U
A
C
U*
U A
G
G
P9.1 G
U
A
G
G
A*
uA C G
P9
A
G*
A
C
G
G
C G
A
s
P9.2
Figure 5. The proposed secondary structure of the intron in the intervening region between yojQ and yojS. The P, Q, R and S in the
box (their exact locations are outlined by the solid lines) indicate highly conserved sequence elements of eukaryotic group I introns.
Conserved base-paired regions are indicated by PI to P9.2. Two arrows indicate putative splice boundaries. AUG and UAG with
asterisks means the putative initiation and stop codon of yojR, respectively.
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p i A-U
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