Download Determination of a 17484 bp nucleotide sequence

Microbiology (1995), 141,3241-3245
Printed in Great Britain
Determination of a 17484 bp nucleotide
sequence around the 39' region of the Bacillus
subtilis chromosome and similarity analysis of
the products of putative ORFs
Eiji Akagawa,' Kenji Kurita,l Takehiko Sugawara,' Kouji Nakamura,'
Yasuyuki Kasahara,2 Naotake Ogasawara2 and Kunio Yamanel
Author for correspondence: Kunio Yamane. Tel./Fax: +81 298 53 6680.
1
Institute of Biological
Sciences, University of
Tsukuba, Tsukuba-shi,
lbaraki 305, Japan
* Graduate School of
Biological Sciences, Nara
institute of Science and
Technology, Ikoma-shi,
Nara 630-01, Japan
We have determined a 17484 bp nucleotide sequence around the 39" region,
located about 480 kb downstream from the zero position of the Bacillus
subtilis chromosome physical map. Among the 17 putative ORFs identified,
orfl and orf2 seem to correspond to mtlA and mtlB, encoding mannitolspecific phosphotransferase enzyme II and mannitol-I-phosphate
dehydrogenase, respectively. O f f 4 seems to be another signal peptidase Igene
(sips) of B. subtilis. The putative products of six ORFs were similar to known
proteins in data banks, namely a hypothetical 29.7 kDa protein of Escherichia
coli (0rf7)#a lactam utilization protein (orf8), the urea amidolyase of yeast
(Orflz), the IclR regulatory protein for aceAB of Salmonella typhimurium
(0rf13)# penicillin-binding protein 2 (Orf16) and aryl-alcohol dehydrogenase
(Orfl7). The amino acid sequence of Orf3 showed 34% identity with that of
LeuC of B. subtilis, though they seem to be functionally different. The
remaining seven ORFs did not show similarity to any known proteins.
Keywords : Bacillus subtilis, genome sequencing, 39" region
The DNA segment of the linking clone pNEXT59 has
been located near the 39" region of the Bacillus szlbtilis
chromosome (Itaya & Tanaka, 1991). The region lies
about 480 kb downstream from the zero position of the B.
szlbtilis physical map and approximately 450 kb downstream from the replication origin, oriC. As part of the B.
szlbtilis genome cooperative project between European
and Japanese laboratories to determine the entire B.
szlbtilis genome nucleotide sequence, we sequenced and
analysed 17484 bp of the 39" region.
Nineteen independent clones containing the pNEXT59
DNA region were isolated by plaque hybridization
(Sambrook e t al., 1989) using 32P-labelledpNEXT59 as a
probe from a ADASHII library of the B. szlbtilis 168 trpC2
chromosome partially digested with San3AI. Among
them, clones 74 and 333 were used for sequence analysis.
An upstream region (region A in Fig. 1) from clone 333
DNA and a downstream region (region B in Fig. 1) from
clone 74 DNA were prepared by means of inverse PCR
..........................................................................................................................................................
Abbreviations: aa, amino acid; SD, Shine-Dalgarno.
The GSDB/EMBUDDBJ/NCBI accession number for the nucleotide sequence
reported in this paper is D38161.
0002-0090 0 1995 SGM
(Ochman e t al., 1988) using the restriction enzyme PstI.
Fig. 1 indicates the DNA fragments sequenced in this
study. Almost all sequences were determined by shotgun
0
I
2
8
I
6
4
1
1
1
10
I
12
I
14
Clone 74
R?2ZZZa
E
t
I
1
P
!
E
EE
II
I
2
3
++-b
P
I
E
I
7
8 9 10 1 1
+++++-b+--+*+
4 5 6
I
pNu(T59
Clone 333
I
16 kb
I
E
E
I
I
12
13 14~'
FB
B
P E
I
16
I
I
17
+
..........................................................................................................................................................
Fig. 1. Restriction map and ORF organization of the B. subtilis
chromosome around the 39" region, located about 480 kb
downstream from the zero point of the physical map of ltaya &
Tanaka (1991). The upper part of this map shows the location
of the cloned DNA in ADASHII clones 333 and 74 that were
hybridized with pNEXT59 and the location of fragments A and
B prepared by means of inverse PCR using the restriction
enzyme Pstl. The restriction sites BamHl (B), EcoRl (E) and Pstl
(P) are indicated. ORFs are indicated by thick arrows and the
arrowheads show the direction of their transcription and
translation.
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3241
E. A K A G A W A a n d O T H E R S
Table 1. Putative ORFs in the 17484 bp sequence around the 39" region of the B. subtilis
chromosome
ORF
Endpoints
(nucleotides)
Size of SD consensus sequence (upper case)
product
and initiation codon (bold)
(aa/kDa)
or-1
orf2
249 > 2078
2134 > 3231
3706 > 4239
4532 > 5092
5399 > 5842
5 848 > 6356
6562 > 7308
7651 > 8148
8350 > 8685
8978 > 9514
9541 > 10311
10358 > 12091
12110 > 12847
12925 > 13563
13755 > 14066
14425 > 16428
16533 > 17432
610165.5
366140.5
178119.7
187121.2
148117.5
170119.5
249128.1
166117.9
112111.7
179118.9
257128.1
578163.8
246127.3
213123.6
104111.8
668174.4
300/34*1
orf3
orf4
Orf5
orj6
KP
orf8
orf9
OrflO
orfll
orfl2
orfl3
orf 14
orfl5
0rfl6
Orfl 7
GGAGGgttta taatg
GAGGTgaactgacatg
atg
AAGGAagttg
AAtAGGAccgggcaggctggcatgtg
AAAGGAGatataaaatg
AAAGaGAGGTacatatatg
AGGAGaTcc t a g t g t t a t g
AAAGcgGGcagc t g g t c a t t g
AAAGGAatcca t t c c g c a g g t g
AAg t GGAGc a a t t a a a caaaatg
AAaGAGGTgagaaagatg
AAacgGGAtgaa tgatg
GGAGGaaagaacatg
atg
AAGGcAGGGaaagtcatg
AAaGAGGga t t t tgcatg
sequencing using a Dye Primer Cycle Sequencing Kit
(Applied Biosystems). The sequences at the gaps and
junction regions of the DNA fragments were determined
using region-specific primers and the Taq DyeDeoxy
Terminator Cycle Sequencing Kit (Applied Biosystems)
as described previously (Ogawa etal., 1995). The compiled
sequences were analysed using BLAST e-mail servers in the
Human Genome Center, Institute of Medical Science,
University of Tokyo, Japan, as described previously
(Ogawa e t al., 1995).
One BamHI, eight EcoRI and three PstI restriction sites
were detected in this DNA region, but there were no BgAI
sites. Putative ORFs in the region were searched in all six
possible translation fr\ames. Based on the length of the
ORF and the presence of a preceding Shine-Dalgarno
(SD) sequence, 17 putative ORFs starting from an ATG,
G T G or T T G initiation codon were identified (Fig. 1 and
Table 1). The initiation codon for 13 of them was ATG,
for orf5 and o r -10 it was GTG and for or-# and or-?)it was
TTG. No suitable SD sequences were identified for or-3
or or-IS.
The orientation of transcription and translation of all 17
ORFs was the same; no sizeable ORF was found in the
opposite orientation.
The amino acid sequence of the putative product of each
ORF was compared with the non-redundant protein data
banks and the results are summarized in Table 2. Ten
ORF products (Orfl, Orf2, Orf3, Orf4, Orfl, Orf8,
Orf12, Orf13, Orf16 and Orfl7) were similar to known
proteins. However, none of the ORFs had sequence
identity with a known B. mbtilis gene.
or-1 encodes a polypeptide of 610 amino acids (aa) and its
sequence showed similarity over the entire length with
3242
those of mannitol-specific phosphotransferase enzyme
I1 (MtlA) of Escbericbia coli (637 aa), and mannitol
transport protein of Bacillus stearotbermopbih (471 aa) and
Stapkylococcus carnosus (505 aa). There are highly homologous regions in the N-terminal 370 aa of the four
enzymes, whereas the aa sequences around position
400-500, corresponding to the middle region of the E. coli
enzyme, differed. However, the identity in the C-terminal
100 aa regions of Orfl and the E. coli enzyme was about
50 %. Therefore, Orfl is the counterpart of mannitolspecific phosphotransferase enzyme I1 in B. subtilis.
Furthermore, the deduced aa sequence of Orf2 (366 aa)
has similarity to those of mannitol-1-phosphate dehydrogenase (MtlD) of E. coli (382 aa) and Streptococctls mgtans
(357 aa). Therefore, or-1 and or-2 in this DNA region are
the counterparts of mtlA and mtlD of E. coli. The two
genes seem to correspond to mtlA, the mutants of which
lack mannitol transport, and mtlB encoding mannitol-1phosphate dehydrogenase of B. stlbtilis, although their
nucleotide sequences have not been determined
(Chaudhry e t al., 1984). The location of these two genes
nearly coincides with the mtlA and mtlB loci on the
genetic map of B. subtilis (Anagnostopoulos e t al., 1993).
The aa sequence of Orf3 comprises 178 residues with low
similarity to the middle region of LeuC (3-isopropylmalate
dehydrogenase, which acts at the third step of leucine
biosynthesis) of B. subtilis. This gene encodes a polypeptide of 365 aa and has been mapped at 247" on the
chromosome. It is therefore unlikely that or-3 encodes
LeuC; it may have another function in B. szlbtilis.
The predicted aa sequence of Orf4 (184 aa) showed 46-2%
identity with that of Sips (184 aa) of B. stlbtilis throughout
the entire length. The sips gene has been mapped at 210"
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The 39" B. szlbti1i.r chromosomal region
Table 2. Similarity of the predicted ORF products to other proteins
Product
Similar protein
Identity observed
BLAST
Reference
score
Orfl
Mannitol transport protein (mtlA) of
B. stearothermophilus
69% in the N-terminal
region, aa 1-375
Mannitol-specific phosphotransferase
enzyme I1 (mtlA) of E. coli
Mannitol-specific phosphotransferase
enzyme I1 ( m t l A ) of StapLylococcus
carnosus
60% in the N-terminal
region, aa 5-367
48% in the middle
region, aa 94-374
Or0
Mannitol-1-phosphate dehydrogenase
of Streptococcus mutans
Mannitol-1-phosphate dehydrogenase
of E. coli
43 % in the middle
region, aa 107-274
49% in the middle
region, aa 170-275
358 Honeyman & Curtiss
(1992)
274 Davis e t al. (1988)
Orf3
LeuC of B. subtilis
34% in the middle
region, aa 202-265
29% in the middle
region, aa 143-266
33% in the middle
region, aa 161-256
37% in the middle
region, aa 200-263
114 Imai et al. (1987)
3-Isopropylmalate dehydrogenase of
Leptospira interrogans
3-Isopropylmalate dehydrogenase of
Lactococcus lactis
3-Isopropylmalate dehydrogenase of B.
coagulans
1332 S. A. Henstra and others
(gp:U18943,
unpublished)
1146 Lee & Saier (1983)
727 Fischer & Hengtenberg
(1992)
183 Ding & Yelton (1993)
168 Godon e t al. (1992)
128 Sekiguchi e t al. (1986)
51 YOin the middle
region, aa 19-133
75% in the middle
region, aa 263-282
326 van Dijl e t al. (1992)
Hypothetical 29-7 kDa protein of E.
coli
Hypothetical protein in PDHA 5'
region (ORF1) of B. stearothermophilus
45 % in the C-terminal
region, aa 180-269
50% in the middle
region, aa 31-84
182 Adachi e t al. (1987)
Orf8
Lactam utilization protein of
Aspergillus nidulans
47% in the N-terminal
region, aa 1-90
236 Richardson e t al. (1992)
Orfl2
Urea utilization protein of yeast
43% in the middle
region, aa 1138-1229
191 Genbauffe & Cooper
(1991)
Orfl3
A regulatory protein (IclR) for aceAB
of Salmonella ophimurium
Acetate operon repressor of E. coli
24% in the C-terminal
region, aa 130-267
25 % in the C-terminal
region, aa 130-267
27% in the middle
region, aa 58-155
147 Galinier e t al. (1990)
Orf4
Signal peptidase I of B. subtilis
Signal peptidase I of E. coli
Orfl
Glycerol operon regulatory protein of
Streptomyces coelicolor
Orfl6
Orfl7
79 March & Inouye (1985)
132 Hawkins e t al. (1990)
146 Sunnarborg e t al. (1990)
85 Smith & Chater (1988)
Penicillin-binding protein 2 (mecA) of
Staphylococcus aureus and S. epidermidis
Penicillin-binding protein 2 of E. coli
46% in the middle
region, aa 320-558
49% in the middle
region, aa 320-397
593 Ryffel e t al. (1990)
Aryl-alcohol dehydrogenase of
Phanerochaete cbrysosporium
39% in the C-terminal
region, aa 223-286
Hypothetical 29.4 kDa protein in the
rmH-dniR intergenic region of E. coli
51 YOin the N-terminal
half, aa 100-132
100 A. Muheim and others
(gp: L08964,
unpublished)
96 K. Nishimura and others
(EMBL D12650,
unpublished)
on the chromosome (Sorokin etal., 1993). The aa sequence
of Orf4 also showed low similarity to signal peptidase I
(Lep) of E. coli, and four highly homologous regions
158 Asoh e t al. (1986)
(A-D) were detected in Orf4, Sips and Lep of E. culi and
Salmonella ty~himurium(Fig. 2). Furthermore, three amino
acid residues, corresponding to Ser43, Lys83 and Asp153
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3243
E. A K A G A W A a n d O T H E R S
t*
Orf4
B. sub
E . coli
S. typ
****
**+
26 :
23:
70:
71:
B
A
*
C
*
**
**
t****
**
D
of Sips, which are critical for signal peptidase activity
(van Dijl e t al., 1995), were also conserved. Therefore, it
is likely that Orf4 is another signal peptidase I of the Sips
type. T o determine how many genes encoding Sips-like
proteins are present in the B. stlbtilis chromosome, we
performed DNA-DNA hybridization experiments. By
comparing the nucleotide sequences between sips and
orf4, we found a highly homologous region between
positions 4742 and 5019. We prepared a 278 bp fragment
by PCR using the DNA of clone 333 as the template and
two specific oligonucleotides as primers. Since this
fragment and its homologue are contained in the 4.5 and
3-6 kb PvdI fragments around orf# and sips, respectively,
Southern hybridization was performed using PvtlIIdigested B. stlbtilis chromosomal DNA and a 32P-labelled
278 bp fragment as probe. Four positive bands (4.5, 3.6,
3.0 and 0.7 kb) were detected. Among them, the 4.5 and
3.6 kb bands gave strong signals while the other two (3.0
and 0.7 kb) were weak (data not shown). These results
indicated that there are at least two signal peptidase I
genes (and probably two more) in the chromosome.
orfl2 and o r -16 may be the genes for urea amidolyase and
penicillin-binding protein 2, respectively. The aa sequence
of Orfl7 showed very low similarity to that of arylalcohol dehydrogenase of Phanerocbaete chrysosporitlm.
The products of orf5, orf6, orf9, o r f l I , orfl4 and orfl.5 were
not similar to any known proteins in the data banks.
ACKNOWLEDGEMENTS
The authors thank D r M. Itaya for providing the Not1 linking
clone pNEXT59, and Ms N . Foster for critical reading of the
manuscript. This research was supported by a Grant-in-Aid for
3244
...................................................... ..............................................
Fig, 2. A comparison of the deduced
amino acid sequences of Orf4 and Sips
(B. sub) of B. subtilis with those of signal
peptidase I of E. coli (E. coli) and
Salmonella typhimurium (5. typ). Dashes
indicate spaces inserted in the sequences
to optimize the alignment. Highly
conserved regions (A-D) in the four
putative signal peptidase I proteins are
underlined.
Amino
acid
residues
common to all four enzymes are
highlighted in black.
Creative Research (Human Genome Project) from the Ministry
of Education, Science and Culture of Japan.
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Received 30 May 1995; revised 21 July 1995; accepted 16 August 1995.
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