Marine Ecosystems Research Department - jamstec japan agency ...

JAMSTEC 2002 Annual Report
Frontier Research System for Extremophiles
study. We have already sequenced , shotgun
clones and a statistical coverage of sequenced region
reached .-fold at the stage of , clones. The
assembly using Phrap yielded contigs, and the
total length of each contig was . Mb, corresponding
to .% of the whole genome of G. kaustophilus
HTA (. Mb).
For finishing the sequencing of the whole genome,
we tried to fill the gaps by using the sequences of both
end of large library insert and by means of PCR with
the primers designed based on the internal sequence in
each contig. Two hundred fifty-seven gaps were
closed by the sequences of reverse side of the shotgun
clones and gaps were bridged by λ phage inserts.
To cover the remaining gaps with PCR product, all
possible pairs of primers were tested whether or not
they gave a PCR product. Sequencing the resulting
PCR products closed the relevant gaps. Although only
one gap which should be closed still remains, the the
genome sequencing project of G. kaustophilus will be
completed soon.
1.4. Diversity and distribution of insertion sequences
among alkaliphilic bacilli
The genome sequences of two terrestrial mesophilic
Bacillus species, alkaliphilic Bacillus halodurans and
neutrophilic Bacillus subtilis, have previously been
published. Through a series of genome analysis studies,
it became clear that the B. halodurans genome
contains fifteen kinds of new insertion sequences
(ISs), IS641 - IS643, IS650 - 658, IS660, IS662, IS663
and a group II intron (Bh.Int) in contrast to no IS element
in the genome of Bacillus subtilis . Out of
ISs identified in the B.halodurans C- genome,
twenty-nine were truncated, indicating the occurrence
of internal rearrangements of the genome. This is one
of the notable features of this genome. Another whole
genome sequence of an extremophilic Bacillus-related
species, Oceanobacillus iheyensis HTE, showing
extremely halotolerant and alkaliphilic phenotypes,
has recently been reported.
Fourteen kinds of the insertion sequences (ISs)
except for IS663, IS641 to IS643, IS650 to 658, IS660,
and IS662, and a group II intron (Bh.Int) identified in
alkaliphilic B. halodurans C- genome were also
detected in other strains of the same species by PCR
and southern blot analysis. The transposase (Tpases) of
IS653 identified in the genomes of strains of B.
halodurans were found to have become most diversified
among all ISs identified in the genomes of
strains. A new IS element designated IS661, with
inverted repeats (IRs) of bp long, was present within
the IS identified in the genome of B. halodurans
A. Also, a new transposon designated Tn3271bh
was identified within the IS642 element in the A
genome, which is a similar transposon identified in
thermophilic Geobacillus stearothermophilus T-. The
new transposon, Tn3271bh generated -bp duplication
of the target site sequence and carries -bp inverted
repeat. On the other hand, fourteen kinds of ISs except
for IS643 and IS658 were distributed in the genome of
obligately alkaliphilic B. alcalophilus. It was found
that most of the ISs identified in the B. halodurans
genome were widely dispersed in the genomes of other
Bacillus species unrelated to the phylogenetic relationship
based on S rRNA sequences.
1.5. Global gene expression profiles of Bacillus halodurans
C-125 grown under alkaline conditions
Recent advance of technology in functional genomic
such as DNA microarray provided a way to understand
the metabolic pathway and genetic expression
patterns in a global genomic scale. Bacillus halodurans
C- is one of the most important alkaliphilic
strains whose whole genome sequence has been determined.
This genomic information made it feasible to
apply the DNA microarray technology for functional
analysis of the genome. B. halodurans C- is a typical
alkaliphile that can grow under alkaline conditions
as well as under neutral conditions. Thus, the strain
B. halodurans C- seems to be a unique organism
to investigate the global gene expression pattern asso-
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