Marine Ecosystems Research Department - jamstec japan agency ...

JAMSTEC 2002 Annual Report
Frontier Research System for Extremophiles
rDNA, internal transcribed spacer, .S rDNA, S
rDNA and elongation facter -. Cryptococcus surugaensis
sp. nov., a novel yeast species from sediment
collected on the deep-sea floor of Suruga Bay was
described.
In , newly isolated microorganisms preserved
in our facility included bacteria strains ( isolates
and others) and yeast strains (from Japan
Trench), and these strains are being stored in liquid
nitrogen conditions. Twenty-two deep-sea sediment
samples obtained by several research vessels in
were also preserved in liquid nitrogen conditions. In
total, we now have types of deep-sea sediment
samples in the liquid nitrogen storage tank.
2.2. Microbial adaptation to high-pressure environments
(a) Identification of the genes responsible for high-pressure
growth in the yeast Saccharomyces cerevisiae
The study aims to establish the molecular basis
responsible for the properties of piezosensitive, piezotolerant
or piezophilic growth in microorganisms and
to identify certain piezosensor (s) of the cell.
We have reported that the availability of tryptophan
is primarily important for high-pressure growth in
S. cerevisiae. During incubation of the wild-type cells
at MPa (approximately atm), the tryptophan
permase Tat is degraded leading to growth arrest.
Overexpression of Tat confers cell growth at this
pressure. Analysis of the high-pressure growth
mutants yielded four linkage groups, that is, HPG1,
HPG2, HPG3 and HPG4. The HPG1 mutation sites
were located in the HECT-domain of the ubiquitin ligase
Rsp. Fig. shows the mutation site within a predicted
structure of the HECT-domain. Rsp is
involved in the intracellular protein degradation
including Tat. The Tat level was indeed enhanced in
the HPG1 mutants at both . and MPa, although
the level was decreased in the wild-type strain at
MPa. The Rsp-binding protein Bul was revealed to
be a negative regulator for Tat under high-pressure
Fig. 4 The predicted structure of the HECT domain of Rsp5 ubiquitin
ligase. The HPG1 and previously known mutation
sites are shown in green. A probable pathway accessible
of E2-bound ubiquitin is shown in orange. Red, α -helix;
Blue, β -sheet.
condition. We have also cloned the HPG2 gene. The
HPG2 was allelic to TAT2 itself. The HPG2 mutation
sites were located in the N- or the C-terminal domain
of Tat and the Tat protein level was enhanced in the
mutants. Taken all results together, we propose a
model for the high-pressure sensing pathway, depicting
that Rsp in combination with Bul regulates Tat
through its N- or C-terminus for degradation in
response to increasing hydrostatic pressure. This is the
first case in which high-pressure response was molecularly
investigated in eukaryotic cells.
(b) Construction of transformants responding to high
hydrostatic pressure in bacteria
The gfp (green fluorescent protein) gene under the
control of a high-pressure inducible-lac promoter was
introduced to the cells of Escherichia coli. Green fluorescence
was indeed detected when the transformant
was grown at MPa (Fig. a). Next, we introduced
the gfp gene under the control of glnA promoter of
Shewanella violacea or cadA promoter of Moritella
japonica to the cells of M. japonica. As a result, green
fluorescence was detected when the transformant was
76