Marine Ecosystems Research Department - jamstec japan agency ...

JAMSTEC 2002 Annual Report
Frontier Research System for Extremophiles
also carried out DNA chip analysis to explore the gene
expression patterns in different concentrations of
NaCl. The results showed that a lot of genes encoding
proteins related to cell membrane and cellular transport
systems were significantly induced by Na + ion.
Another characterisitic of B. halodurans C- is
the alkali-dependent swimming motility. This alkalidependent
swimming motility was not found from
other alkaliphilic Bacillus such as Oceanobacillus
iheyensis HTE. We made several mutants that
showed alkali-independent swimming motility, and
then systematically examined the mRNA profiles with
the DNA chips. Significant alterations of several
genes encoding proteins such as flagellin and glycolate
oxidase were found between wild type and
mutant. It was suggested that these genes may be
involved in alkali-dependent swimming motility.
1.6. Proteomic analysis of pH-dependent gene
expression in facultative alkaliphilic Bacillus
halodurans C-125
The complete identification of coding sequences in
a number of model species has led to announce the
beginning of the post-sequencing era; rapid advances
in genomic sequencing technology and bioinformatics
have established the field of genomics to investigate
the expression profile of the genes identified in the
genome through mRNA display. Since recent studies
have demonstrated, however, a lack of correlation
between the transcriptional profiles and the actual protein
levels in the cells, the comprehensive analysis of
the gene products is indispensable to link genomic
data to biological function. Analysis of the genome of
Bacillus halodurans C- was initiated in as a
standard model for facultative alkaliphilic Bacillus
strains, and the systemic sequencing of the whole
genome of B. halodurans C- finished in . The
B. halodurans C- is the first industrial strain whose
whole genomic sequence has been determined.
The objective of proteomics is a large-scale, comprehensive
characterization of the proteins and protein
interactions in the cells. The proteomic analysis with
sensitive mass spectrometry subsequent to high-resolution
two-dimensional electrophoresis (-DE) was
used to identify the proteins involved in the mechanisms
of adaptation to alkaline environment in
B. halodurans C-. The total proteins and membrane
protein fractions of the B.halodurans C-
cells grown to the mid- exponential phase at pH .
and pH . were subjected to -DE. A pH gradient of
to was chosen for the isoelectrofocusing (IEF) due
to the stability and reproducibility of the gradient in
this pH range. The results of the -DE are shown in
Fig.. Approximately protein spots could be
resolved by LabScan v. software (Amersham
Biosciences) on a gel stained with Coomasie Brilliant
Blue (CBB) R-. The amounts of the protein in
spots out of the were more than twofold higher in
pH. culture than pH . cultures. All proteins
were identified by N-terminal amino acid sequencing
and LC/MS/MS. Eight proteins of interest were
pI
kDa
4.0
94 A
67
43
30
20
14
94
67
43
30
20
14
C
7.0 4.0 7.0
Fig. 3 Two-dimensional gel electrophoresis of B. halodurans
C-125 mid-exponential phase cell proteins. A total of 150
µg of total proteins (A, pH 7.0 and B, pH10.0) and membrane
proteins (C, pH 7.0 and D, pH10.0) were loaded for
each gel. Prior to electrophoresis, samples were incubated
in SDS buffer at room temperature for 15 min. After
electrophoresis, gels were stained with Coomassie blue R-
250. Spots whose density was increased in the growth of
pH 10.0 are marked with circles.
B
D
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