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Whole genome sequencing
project of alkaliphilic Bacillus halodurans C-125
- What is alkaliphilic Bacillus species ?
Generally, alkaliphilic Bacillus strains
cannot grow or grow poorly under neutral pH conditions, but grow
well at pH higher than 9.5. Since 1969, we have isolated a great
number of alkaliphilic Bacillus strains from various environments
and have purified many alkaline enzymes. Actually, as mentioned
above, alkaliphilic bacteria containing Bacillus strains
have been isolated from even deep-sea sediment collected at ranging
1,000 to 10,000 m depth at a frequency of 0.2 x 102
to 2.3 x 104 per colonies g-1 dry sea mud.
Over the past two decades, our studies have focused on the enzymology,
physiology, and molecular genetics of alkaliphilic microorganisms
to elucidate their mechanisms of adaptation to alkaline environments.
Industrial applications of these microbes have also been investigated
and some commercial enzymes from alkaliphilic Bacillus
strains have brought great advantages to industry. Thus, it is
clear that alkaliphilic Bacillus strains are quite important
and interesting not only academically but also industrially.
An alkaliphilic bacte-rium, strain C-125 (JCM9153), isolated
in 1975, was identified as a member of the genus Bacillus
and reported as a b-galactosidase
and xylanase producer. It is the most thoroughly characterized
strain, physiologically, biochemically, and genetically, among
those in our collection of alkaliphilic Bacillus isolates.
Recently, this strain was reidentified as Bacillus halodurans
based on phylogenetic analysis using 16S rDNA sequence and DNA-DNA
hybridization analysis.
- Genome analysis of alkaliphilic Bacillus halodurans C-125
A physical map of the chromosome of this strain
was constructed to facilitate further genome analysis and the
genome size was revised from 3.7 Mb to 4.25 Mb. Complete digestion
of the chromosomal DNA with two rare cut restriction endonucleases,
AscI and Sse8387I, each yielded 20 fragments ranging
in size from 20 to 600 kb. Seventeen linking clones were isolated
in each instance to join the adjacent AscI or Sse8387I
fragments in the chromosomal map. All AscI linking clones
isolated were sequenced and analyzed by comparison with the BSORF
database to map the genes in the chromosome of strain C-125.
Several ORFs showing significant similarities to those of B.
subtilis in the AscI linking clones were positioned
on the physical map. The oriC region of the C-125 chromosome
was identified by Southern blot analysis with a DNA probe containing
the gyrB region.
Seventeen Sse8387I linking clones isolated
from the chromosome of Bacillus halodurans C-125 for the
purpose of constructing a physical map were sequenced and analyzed
by compari-son with the BSORF database and the non-redundant
protein databank. The orientations of Sse8387I or AscI
linking clones serving to join adjacent fragments were determined
by Southern blot analysis using specific DNA probes. One-third
of the open reading frames (ORFs) identified in the Sse8387I
linking clones showed no significant similarity to any protein
so far reported. The ORFs showing significant similarities to
those of Bacillus subtilis were mapped in the chromosome
of strain C-125 and the locations of the putative genes on the
map differed completely from those of B. subtilis orthologues.
The nucleotide sequences of three independent
fragments (designated No. 3, 4, and 9; each 15 - 20 kb in size)
of the genome of alkaliphilic Bacillus sp. C-125 cloned
in a l phage vector have been determined.
Tirteen putative open reading frames (ORFs) were identified in
sequenced fragment no. 3 and eleven ORFs were identified in no.
4. Twenty ORFs were also identified in fragment no. 9. All putative
ORFs were analyzed in comparison with the BSORF database and
non-redundant protein databases. The functions of 5 ORFs in fragment
no. 3 and 3 ORFs in fragment no. 4 were suggested by their significant
similarities to known proteins in the database. Among the 20
ORFs in fragment no. 9, the functions of 11 ORFs were similarly
suggested. Most of the annotated ORFs in the DNA fragments of
the genome of alkaliphilic Bacillus sp. C-125 were conserved
in the Bacillus subtilis genome. The organization of ORFs
in the genome of strain C-125 was found to differ from the order
of genes in the chromosome of B. subtilis, although some
gene clusters (ydh, yqi, yer, and yts)
were conserved as operon units the same as in B. subtilis.
- Whole genome shotgun sequencing of
Bacillus halodurans
Systematic sequencing of the whole genome
of Bacillus halodurans C-125 was routinized at the beginning
of May in 1998 and has been done in the middle of August.
Chromosomal DNA was isolated from Bacillus
halodurans C-125 as described above. A 20mg
aliquot of chromosomal DNA was sonicated for 5 to 25 sec with
a Bioruptor UCD-200TM. The sonicated DNA fragments were blunt-ended
using a DNA blunting kit and fractionated by 1% agarose gel electrophoresis.
DNA fragments 1-2 kb in length were excised from the gel and
eluted by the freeze-squeeze. The DNA recovered was ligated to
the SmaI site of pUC18, and introduced into competent XL1-Blue
cells. We usually could obtain transformants with a frequency
of 5 - 6 x 105 per mg-DNA
and they were used for amplification of insert by using colony
PCR method.
The DNA fragment inserted into pUC18 was amplified
by PCR using M13-20 and reverse primers. PCR fragments, treated
with Exonuclease I and Shrimp alkaline phosphatase to eliminate
excess primers in the PCR reaction mixture were used for sequencing
analysis as template DNA. Sequencing was performed with a DNA
sequencer ABI PRISM 377 using a Taq Dye Terminator Cycle Sequencing.
We sequenced 60,000 shotgun clones and a statistical coverage
of sequenced region reached 10-fold at this stage. DNA sequences
determined by means of the ABI sequencer were assembled into
contigs using Phrap (http://bozeman.mbt.washington. edu/phrap.docs/phrap.html)
with default parameters and without quality scores.
Now we are preparing to open all sequence
data and they will appear on the web site in next spring.
We developed a semi-automated genome analysis
system called Genome Gambler in order to support the current
whole-genome sequencing project focusing on alkaliphilic Bacillus
halodurans C-125. Genome Gambler was designed to reduce
the human intervention required and to reduce the complications
in annotating thousands of ORFs in the microbial genome. Genome
Gambler automates three major routines: analyzing assembly results
provided by genome assembler software, assigning ORFs, and homology
searching. Genome Gambler is equipped with an interface for convenience
of annotation. All processes and options are manipulatable through
a WWW browser that enables scientists to share their genome analysis
results without choosing computer platforms. The GAMBLER software
will be released through Mitsui Knowledge Industry Co. Ltd. (MKI,
http://bio.mki. co.jp/) at the beginning of 2000. Software maintenance,
bug fixes, and upgrades will be handled by MKI as well. The Genome
Gambler software is now available at no charge to academic users
for research purposes.
1. Kaoru Nakasone, Yoshihiro Takaki, Hideto Takami,
Akira Inoue and Koki Horikoshi (1998) Cloning and expression
of the gene encoding RNA polymerase a subunit from alkaliphilic
Bacillus sp. strain C-125. FEMS Microbiol. 168,
269-276
2. Hideto Takami, Kaoru Nakasone, Chie Hirama, Noriaki
Masui, Fumie Fuji, Yoshihiro Takaki, Yuka Nakamura, Akira Inoue,
and Koki Horikoshi (1999) An improved physical and genetic map
of the genome of alkaliphilic Bacillus sp. C-125. Extremophiles
3, 21-28
3. Hideto Takami, Kaoru Nakasone, Naotake Ogasawara,
Yuka Nakamura, Chie Hirama, Fumie Fuji, Yoshihiro Takaki, Noriaki
Masui, Akira Inoue, and Koki Horikoshi (1999) Sequencing of three
lambda clones from the genome of alkaliphilic Bacillus
sp. C-125. Extremophiles 3, 29-34
4. Hideto Takami, Yoshihiro Takaki, Kaoru Nakasone,
Chie Hirama, Akira Inoue and Koki Horikoshi (1999) Sequence analysis
of 32 kb fragment including the major ribosomal protein gene
clusters in alkaliphilic Bacillus sp. strain C-125.
Biosci. Biotech. Biochem. 63, 452-455
5. Hideto Takami, Noriaki Masui, Kaoru Nakasone, and
Koki Horikoshi (1999) Replication origin region of the chromosome
of alkaliphilic Bacillus halodurans strain C-125.
Biosci. Biotech. Biochem. 63, 1134-1137
6. Hideto Takami, Yoshihiro Takaki, Kaoru Nakasone, Tokuki
Sakiyama, Go Maeno, Rumie Sasaki, Fumie Fuji, Chie Hirama and
Noriaki Masui (1999) Genetic analysis of the genome of alkaliphilic
Bacillus halodurans C-125. Extremophiles 3,
227-233
7. Hideto Takami, Yuichi Nogi, and Koki Horikoshi (1999)
Reidentification of keratinase- producing facultatively alkaliphilic
Bacillus sp. AH-101 as Bacillus halodurans. Extremophiles
3, 293-296
8. Hideto Takami and Koki Horikoshi (1999) Reidentification
of facultatively alkaliphilic Bacillus sp. C-125 to Bacillus
halodurans. Biosci. Biotech. Biochem. 63, 943-945
9. Hideto Takami (1999) New Approaches and Future Scope
for Deep-sea Microbiology: Genome Analysis of Facultatively Alkaliphilic
Bacillus halodurans C-125. In: Extremophiles in the deep-sea
environments (Horikoshi, K and Tsujii K., eds.) Springer-verlag
249-284
10. Hideto Takami, and Terry Krulwich (2000) Re-identification
of facultatively alkaliphilic Bacillus firmus OF4 as Bacillus
pseudofirmus. Extremophiles 4, 19-22
11. Tokuki Sakiyama, Hideto Takami, Naotake Ogasawara,
Satoru Kuhara, Kosuke Doga, Tokio Kozuki, Akira Oyama and Koki
Horikoshi (2000) An automated system for genome analysis to support
microbial whole-genome shotgun sequencing. Biosci. Biotech.
Biochem. 64 (3), 670-673
12. Kaoru Nakasone, Noriaki Masui, Yoshihiro Takaki,
Rumie Sasaki, Go Maeno, Tokuki Sakiyama, Chie Hirama, Fumie Fuji
and Hideto Takami (2000) Characterization and comparative
study on the rrn operons of alkaliphilic Bacillus
halodurans C-125. Extremophiles 4, 209-214
13. Hideto Takami and Koki Horikoshi (2000) Genome
analysis of alkaliphilic Bacillus strain from an industrial
point of view. Extremophiles 4, 99-108
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