FEMS Microbiology
Letters 126 (1995) 159-164
Cell-water deficit regulates expression of rpoC1 C2
( RNA polymerase) at the level of mRNA in desiccation-tolerant
Nostoc commune UTEX 584 ( Cyanobacteria)
Wen-Qin Xie, David Tice, Malcolm Potts
*
Department of Biochemistry and Anaerobic Microbiology, Virginia Tech., Blacksburg, VA 24061, USA
Received 5 October 1994; accepted 15 December
1994
Abstract
Immobilization and short-term air-drying of the cyanobacterium Nostoc commune strain UTEX 584 leads to a complete
depletion of its cellular rpoc2C2 mRNA pool. This mRNA is required for the synthesis of the y and /3’ subunits of
DNA-dependent RNA polymerase (RNA-P). In contrast, RNA-P remains stable in cells during long-term desiccation as
judged from immunoblotting analyses of protein extracts using RNA-P core-specific antibodies. The data indicate that the
extant RNA-P holoenzyme in air-dried cells drives the rapid de novo transcription of rpoClC2 that ensues in response to
cell rehydration.
Keywork
Desiccation tolerance; Cyanobacteria; Nostoc commune; RNA polymerase; Gene expression
1. Introduction
The removal of cell-bound water through air-drying, and the addition of water to air-dried cells,
exercise marked effects on the distribution and activities of bacterial communities. Yet desiccation, as a
major stress parameter in nature, has continued to
escape critical attention and the mechanisms that
contribute to the desiccation tolerance of prokaryotic
cells remain obscure [l].
Of those microorganisms that express a capacity
to tolerate extremes of water deficit, many cyanobacteria - and one form in particular, Nostoc commune
- have a marked tendency
* Corresponding
to do so [2]. N. commune
we have commenced
structural analyses of gene
products synthesized by N. commune strain UTEX
584 (Nostoc 584). This strain has provided a conve-
author.
037%1097/95/$09.50
0 1995 Federation
SSDI 0378-1097(95)00004-6
becomes visually conspicuous in terrestrial limestone
regions, especially those where there is a limited and
variable availability of water [3]. Here, colonies of
this cyanobacterium are subjected to intermittent
wetting and often extended periods of desiccation.
Air-dried cells of N. commune can maintain their
viability despite decades of storage in the air-dried
state [4]. A principal consideration in any appraisal
of the resilience of these cells is the stabilities of
their proteins. Do desiccated cells provide an environment that is conducive to the enhancement of
protein stability, or do tolerant cells accumulate proteins that are inherently more stable than those found
in sensitive cells? To begin to answer these questions
of European
Microbiological
Societies. All rights reserved
160
W.-Q. Xie et al. /FEMS
Microbiology
nient model to study the consequences of air-drying
at the molecular level [4-111. The present account
describes our analysis of the turnover of DNA-dependent RNA polymerase (RNA-P) in cells of Nostot 584 subjected to different cell-water deficits.
The stoichiometry
of eubacterial
RNA-P
is
[ PPa& [a. 1n contrast, the cyanobacterial enzyme contains an additional subunit, y, and has the
stoichiometry
[ @~‘az]a
[13]. Cloning and sequence analysis of the genes from Nostoc 584 that
encode the p (rpoZ31, y (rpoC1) and p’ (rpoC2)
subunits of its RNA-P indicated that y and p’
correspond to the N-terminal and carboxy-terminal
regions of eubacterial p’, respectively [14]. Furthermore, Nostoc 584 rpoC1 and rpoC2 are transcribed
separately from rpoL? as a single, 5.6-kb dicistronic
message (Fig. lA, [15]).
RNA-P is the pivotal component of the transcription apparatus. If cells are to recover from desiccation, either their complement of RNA-P must remain
intact or their pool of rpo mRNA transcripts must
remain intact. Successful recovery with the former
stipulation requires that cells retain intact DNA templates, ancillary transcription factors and a pool of
ribonucleoside
triphosphates.
De novo translation
with the latter stipulation requires that the cells retain
active ribosomes, charged tRNAs and ancillary translation factors. To understand which of these sets of
conditions may prevail we subjected cells to different water stresses, studied the turnover of rpoClC2
mRNAs, and monitored the fate of the subunits of
RNA-P as intracellular markers for the holoenzyme.
2. Materials
and methods
2.1. Growth and immobilization
of cells
Cells of Nostoc 584 were grown as described [6]
and were harvested in the mid-log phase of growth
by centrifugation. The cell pellets were divided into
portions of approximately 0.7 g (wet weight) which
were then spread evenly, as pastes, over the surface
of inert nylon meshes [9]. The cells were incubated
in an atmosphere with a matric water potential (W,)
of -99.5 MPa and were allowed to dry, under a
continuous incident photon flux density of approximately 50 pmol photons m-* s-r at the surface of
Letters I26 (1995) 159-164
the culture vessels, for periods between 1 and 5 days.
These conditions are comparable to those sometimes
experienced by colonies of N. commune growing in
situ. Water loss from colonies ceased after 5 days of
drying at which time the colonies were judged to be
desiccated. Desiccated cells were rehydrated when
necessary through the addition of sterile BGll,
medium [ 161.
2.2. Isolation and purification
of mRh?A
The method of Xie and Potts [15] was used to
purify the intracellular mRNA pools of the air-dried
cells as well as air-dried cells that had been rehydrated for 5 min, 10 min, 30 min, 60 mitt, 24 h or 4
days. RNA pools were extracted from Nostoc 584
cells and were transferred to nylon sheets as described following resolution in 1% (w/v> glyoxal
agarose gels [15]. An 878-b riboprobe (antisense),
complementary to bases 1241 to 2119 of rpoC2, was
synthesized from a deletion plasmid [17] using T7
RNA polymerase in the presence of [35SlUTP (1284
Ci mmol- ‘, New England Nuclear). Nylon membranes were prehybridized in a plastic bag with RNA
hybridization
buffer (5 X SSC, pH 7.0, 50 mM
NaPO,, pH 6.5, 0.5% w/v SDS, 4.5 X Denhardt’s
solution and 55% w/v formamide) at 50°C for 4-5
h. The prehybridization
buffer was then discarded
and the bag was refilled with fresh buffer containing
the 35S-labelled riboprobe. Hybridization
was performed at 50°C overnight, with gentle shaking. After hybridization,
the membrane was rinsed in 1 X
SSC/O.l%
(w/v) SDS buffer for several minutes
with one buffer change. The filter was then washed
in 0.1 X SSC/O.l% (w/v) SDS buffer at 60°C for
50 min, with two buffer changes. Finally, the membrane was rinsed with 1 X SSC/O.l%
(w/v) SDS
buffer and dried. RNA-RNA
hybrids were visualised by autoradiography and the signals were quantified using scanning densitometry (Shimadzu). The
data presented here are representative of those obtained in multiple trials.
2.3. Isolation of proteins and Western blotting
Replicate samples were used to obtain extracts of
total cell proteins in Laemmli buffer [18] using the
methods of Hill et al. [4]. Cells were first frozen in
W.-Q. Xie et al. / FEMS Microbiology Letters 126 (1995) 159-164
liquid nitrogen, ground to a powder in a chilled
mortar and then transferred to 15ml tubes. 2 ml of
Laemmli buffer [18] were added and the mixture was
sonicated (Fisher sonic dismembranator with microprobe) for three consecutive periods of 30 s each, at
a setting of 30. The efficiency of breakage of cells
was monitored using light microscopy. Cell debris
was removed by centrifugation at 35000 rpm in a
Beckman Ti50 rotor for 1 h, at 4°C. The proteins
from equivalent amounts of cell lysate were resolved
161
in 8% (w/v> polyacrylamide gels and were processed for SDS-PAGE and Western blotting as described [19].
3. Results
rpoClC2 transcripts were not detected in RNA
preparations from cells that had been immobilized
and dried in air for 24 h - a period of time that does
core enzyme structure
P
Y
rpoB
B’
rpoC1
W
3.1 kbp
rpocz
5.6 kbp
)
A
abc
d
e
f
-6.6Kb
B
abcde
Fig. 1. (A) Organization of the rpol3 and rpoCIC2 operons and structure of the core enzyme of Nostoc UTEX 584 RNA polymerase.
Straight arrows denote the direction of transcription and numbers indicate the sizes of the transcribed rpo DNA fragments. Bent arrows
signify the positions of characterized promoters. The hatched line indicates the region of rpoC2 that was used to generate a riboprobe. (B)
rpoClC2 transcripts are degraded during air-drying of cells and then are rapidly synthesized upon cell rehydration. Autoradiograph
of a
Northern blot after probing equivalent amounts of RNA pools from cells that had been: a, 1 day dry; b, 5 min rewet; c, 10 min rewet; d, 30
min rewet; e, 60 min rewet; f, control (liquid culture). The position of the 5.6-kb rpoCIC2 transcript is indicated (see A). (C) The a
subunit, a marker for the core RNA-P, remains stable in cells subjected to different water deficits. Western blot of equivalent amounts of
protein extracts from cells that had been: a, 1 day dry; b, 5 days dry; c, 10 min rewet; d, 30 min rewet; e, 60 min rewet; f, 5 h rewet; g, 24 h
rewet; h, 4 days rewet; i, control (liquid culture). Numbers indicate and marked the positions and sizes of molecular mass markers.
162
W.-Q. Xie et al. /FEMS
Microbiology
not lead to desiccation (Fig. 1B). Resolution of
greater amounts of RNA through the overloading of
gels, as well as the implementation
of increased
exposure times during autoradiography, failed to detect any rpoClC2 transcripts in these preparations.
In cells that had been dried for longer periods and
which were desiccated, rpoC1 C2 mRNA was first
detected in extracts from cells that had been rewetted
for 10 min and an extractable pool of rpoClC2
mRNA, equivalent in size to approximately 70% of
the pool present in control cells (in liquid culture),
was present after 60 min of rewetting (Fig. 1B). We
confirmed that the water status of the cells did not
interfere with the efficiency of extraction of their
RNA pools. The amounts and quality of rRNA present in control cells, and in cells that had been dried
for different time intervals, were judged to be equivalent following spectrophotometric
measurements,
after analysis by agarose gel electrophoresis,
and
after probing Northern blots with an Escherichia
coli rRNA probe ([20]; data not presented).
To assess the effects of water deficit on the
protein complement of dried cells their protein extracts were probed with antibodies specific for the
core of RNA-P. Antiserum raised against the core
RNA-P of Anabaena
sp. strain PCC 7120 gave a
pronounced reaction against the c~ subunit of the
Nostoc 584 RNA-P. These experiments
demonstrated that there was no noticeable change in the
relative amounts of the RNA-P a subunit in cells
that were subjected to a range of different water
deficits in comparison to the level of (Y in cells
grown in liquid culture (Fig. 1C). The antiserum
gave weaker reactions with the other components of
the core enzyme as has been observed with the Rpo
proteins of other strains of cyanobacteria and as is
documented
in Fig. 2 of ref. [21]. Nevertheless,
protein extracts from air-dried cells, from rehydrated
cells, and from cells grown in liquid culture (control),
generated signals from the y, /3 and p’ subunits of
the Nostoc 584 core RNA-P of equivalent intensity
(data not shown).
4. Discussion
Cells of Nostoc 584 lose the capacity to maintain
a pool of rpoC1 C2 mRNA following their immobilisation and short-term drying, air-dried cells lack any
Letters 126 (1995) 159-164
detectable rpoCIC2 mRNA, and there is a conspicuous rise in the pool size of rpoClC2 mRNA upon
rehydration of cells. It is possible that Northern
analyses may have failed to identify very scarce
transcripts. However, previous studies have shown
that there is a loss of integrity of nucleic acids in
desiccated cells, RNA preparations from cells subject
to long-term
desiccation
are poorly resolved by
agarose gel electrophoresis,
and RNA preparations
from such cells fail to drive in vitro translation
[10,22]. These data are consistent with a mode of
action where RNA-P remains stable, and functional,
in desiccated cells and is responsible for the very
rapid rise in the rpoClC2 pool following subsequent
rehydration. Confirmation
of this latter fact is the
finding that the (Y subunit of RNA-P - a marker of
the presence and the abundance of the core enzyme
in cells -- as well as the other subunits of the core,
were present in all of the Nostoc 584 extracts in
approximately
equivalent
amounts (see Fig. 10.
Prokaryotic cells may contain up to around 10000
copies of RNA-P [23] so it is conceivable that only a
fraction of those copies present in desiccated cells
remains functionally active and capable of initiation
of transcription
upon cell rehydration. These data
indicate that rpoClC2 gene expression is regulated
by water stress at the level of mRNA and the rise in
the rpoC1 C2 mRNA pool size upon cell rehydration,
i.e. de novo transcription, is at the expense of extant
RNA-P holoenzyme. It is not possible to state, at this
point, whether the regulation is at the level of mRNA
stability or rpoClC2 transcription.
Irrespective of the quantities of transcriptionally
active RNA-P in rehydrating cells there is a net
requirement for intact DNA templates. The rapid rise
in the rpoClC2 mRNA pool upon rehydration implies a priori that intact rpoClC2 DNA is available,
but because our previous studies have shown that the
genomic DNA of desiccated Nostoc 584 is subject to
a substantial degree of light-dependent
nicking the
rpoClC2 transcription described here may, in fact,
derive from DNA that has undergone rapid repair
during cell rehydration [lo]. The present data, and
previous observations
that show the intracellular
RNA pools from desiccated N. commune cannot
drive in vitro translation, show that the drying of a
cell leads to a general depletion of its pool of
mRNAs.
W.-Q. Xie et al. / FEMS Microbiology Letters I26 (1995j 159-164
Desiccated cells of N. commune contain massive
amounts of a highly stable (several decades) water
stress protein (Wsp) that is implicated in the modification and/or synthesis of UV-A/B-absorbing
pigments [4]. Enzymes of lipid biosynthesis
remain
functional in these cells as evidenced by an instantaneous onset of lipid biosynthesis upon rewetting [ 111.
At least one secreted enzyme, a protein tyrosine
phosphatase, retains its phosphomonoesterase
activity following drying and rewetting [24,25], and the
Fe protein of nitrogenase remains structurally intact
in cells following their storage for more than a
decade in the air-dried state [5]. If protein stability is
a key to the survival of desiccation-tolerant
cells,
then what is the basis for the stability? Those proteins from extremophiles that have been studied show
no obvious primary sequence divergence from the
corresponding proteins found in mesophiles [26]. N.
commune places a considerable metabolic investment in its secreted UV-absorbing pigments, secreted
Wsp proteins and a secreted extracellular
glycan
[27]. These components may contribute to, and form
part of, an environment,
a protective environment,
that is conducive to the maintenance of protein stability.
Acknowledgements
We thank R. Haselkorn for his kind gift of antiserum. This study was supported by NSF Grant DCB
9103232.
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