Marine Ecosystems Research Department - jamstec japan agency ...

Japan Marine Science and Technology Center
Frontier Research System for Extremophiles
analyses based on each Bacillus-related species
although the genome size of O. iheyensis is kb
smaller than that of the other two Bacillus genomes.
In addition, orthologous relationships emerged in the
comparison with all combinations among the five
genomes used in this study (Fig. ). The putative
proteins characterized on the basis of orthologous relationships
were assigned to the functional categories
used for B. subtilis.
Out of putative proteins without orthologous
relationship to other Gram-positive species,
were orphans showing no significant similarity
of amino acid sequence to any other protein and
were conserved proteins in other organisms. One
hundred seventy-four were conserved proteins of
unknown functions among conserved proteins.
Sixty proteins were grouped into transport/binding
proteins and lipoproteins, which was numerically
the most abundant category. Nearly half of these
proteins are ABC transporter-related proteins. Many
of the orthologs, which were shared between two to
four species in the comparisons were also grouped
into this category, indicating that some of these
transport-related proteins are part of distinguishing
characteristics for subsets of Gram-positive bacteria.
1.2. Construction of the sequence database specifically
for the O. iheyensis genome
We attempted to construct a new database specifically
for the O. iheyensis sequences "ExtremoBase"
as well as the case of B. halodurans because the
genome sequence data should be open to the public
at the same time as the publication of the paper.
We prepared a useful data search system to the
O. iheyensis genome sequence and set up a new
server system for ExtremoBase at Yokohama
Campus of JAMSTEC. ExtremoBase has been
accessible through the World Wide Web server at
http://www.jamstec.go.jp/jamstec-e/bio/jp/topj.html.
1.3. Genome sequencing of Geobacillus kaustophilus
HTA426
The microbial genome is primarily sequenced by
the whole genome random sequencing method, which
is composed of two steps. The first step of this method
is random shotgun sequencing and the second one is
gap filling occurred by assembly of the shotgun clones
sequenced at the first step. Although the shotgun
sequence step is generally performed at a level of -
fold genome coverage, several hundred gaps still
remain at this stage because the random shotgun
library does not contain all clones to cover whole
genomic sequences. Thus, the completion of the
genome sequencing project depends highly on the gap
filling process.
To raise efficiency of the gap filling, we attempted
to improve the ways for construction of the shotgun
library and sample treatment for sequencing. First, the
temperature condition for ligation reaction was optimized
to obtain a high frequency of transformation.
By lowering the reaction temperature from the usual
˚C to ˚C, the transformation efficiency (. x
transformants/g DNA) increased approximately
- to -fold higher than the usual result. Secondly,
the purification condition for PCR product sequenced
was optimized to raise the success rate of the sequencing.
Total amount of PCR product was usually treated
twice with Endonuclease I (Exo I) and Shrimp
Alkaline Phosphatase (SAP) to remove excess primer.
In this study, only the amount of PCR product needed
for sequence reaction was treated with higher concentration
of Exo I and SAP. Consequently this method
shortened the time for sample treatment, and also
increase the success rate for sequencing from %
to %.
The genome of Geobacillus kaustophilus HTA
was primarily sequenced by whole genome random
shotgun method as well as other microbial genome
sequencing project. The random shotgun library with
an average insert of . kb or kb was constructed
from the genomic DNA by improved methods in this
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