FEMS Microbiology Letters 15 (1982) 249-252
Published by Elsevier Biomedical Press
249
Some physiological features of a Halococcus sp. at low salt
concentrations
F. Rodriguez-Valera, A. Ventosa, E. Quesada * and F. R u i z - B e r r a q u e r o
Department of Microbiology, Faculty of Pharmacy, Sevilla, and * Department of Microbiology, Faculty of Pharmacy, Granada, Spain
Received 28 May 1982
Accepted 3 June 1982
1. I N T R O D U C T I O N
Extremely halophilic bacteria were considered
to be adapted for living in highly concentrated salt
solutions. In fact, the two genera described Halobacterium and Halococcus require high concentrations of salt to grow, usually more than 15%(w/v)
NaC1 [1]. Furthermore, Halobacterium cells lyse
when the salt concentration is lower than 10%(w/v)
although this limit can be lowered in the presence
of Mg 2+. However, Halococcus cells withstand
very low salt concentrations [2]. In a previous
publication we described the isolation of this kind
of microorganisms from the Mediterranean Sea
and showed that Halococcus cells can stay alive for
very long periods suspended in sterile seawater [3].
One question raised by these results is how this
extreme halophile manages to survive under these
conditions. The ability to survive in seawater could
play an important role in the dispersion of these
microorganisms into newly formed hypersaline
media. To determine if the halococci remain able
to develop any metabolic activity at seawater salt
concentrations we have measured their respiration
rate under these conditions, and their ability to
take up radioactively labelled leucine. We have
also measured the amount of cell-associated K +
that is retained at seawater salt concentrations.
Since this is the compatible solute of extreme
halophiles [1] it is interesting to know if it could
reach the low levels that theoretically should correspond to the seawater salt concentration.
2. M A T E R I A L S A N D M E T H O D S
The strain Halococcus sp. NCMB757 was used.
The cells were grown in liquid medium (100 ml in
500-ml erlenmeyer flasks) with lateral agitation
(100 strokes/min). The medium contained 25%
( w / v ) salts and 0.5% ( w / v ) yeast extract (Difco)
and was adjusted to p H 7.0. For all experiments
we have used a mixture of salts keeping the same
proportions as seawater salts and in sufficient
amounts to give the final total salt concentration
indicated as described previously [4]. The flasks
were incubated at 41°C for 2 weeks.
2.1. Transport experiments
The cells were washed with 25% ( w / v ) salt
solution and then a suspension with an absorbance
of 0.5 was prepared. This washed suspension was
then added to 4.5 ml of sterile 25, 15, 3 or 0.5%
( w / v ) salt solution in 25-ml erlenmeyer flasks. To
measure the amino acid uptake, 0.01 ml of a
solution of [14C]leucine (10 m C i / m m o l ) was added to give a final concentration of 10-aM.
[14C]Leucine was furnished by New England
Nuclear Corporation, Boston, MA, U.S.A. The
0378-1097/82/0000-0000/$02.75 ~:~ 1982 Federation of European Microbiological Societies
250
mixture was incubated at 41°C in a bath with
rotatory shaking, 0.1-ml samples were withdrawn
every 10 rain and filtered through 0.45 # m membrane filters which, after extensive washing with
25% ( w / v ) salts, were placed in plastic scintillation
vials to which were added 10 ml of a cocktail
consisting of dioxane with 10% ( w / v ) naphthalene
and 0.5% ( w / v ) diphenyloxazole. Samples were
counted overnight in a Beckman LS-100C liquid
scintillation counter. All uptake measurements
were corrected for retention of the 14C-labeled
amino acid by the filters in zero-time controls.
2.2. Measure of cell-associated potassium
Cells were grown as described above. Once an
absorbance of 1.0 was reached by the culture, the
cells were harvested by centrifugation, washed with
25%(w/v) salt solution and finally resuspended in
an equal volume of 0.5, 3, 15 or 25%(w/v) salt
solution in 500-ml erlenmeyer flasks that were
then incubated at room temperature in the dark
and with slow lateral shaking (to keep the cells
suspended) for a week. After this time, the suspensions were centrifuged, washed twice with a pure
NaC1 solution of the same concentration as the
suspension solution. Finally, the pellet was resuspended in 10 ml distilled water. The K + concentration of this suspension was determined by
atomic absorption, with a Perkin-Elmer 373 atomic
absorption spectrophotometer. The protein content was determined by the Lowry method.
3. RESULTS A N D D I S C U S S I O N
In Fig. 1 the result is shown of a typical experiment of transport of a radioactively labelled amino
acid by the strain of Halococcus utilized. The
amino acid was transported into the cell at all the
salt concentrations used and only with 0.5%(w/v)
salts was there an undetectable uptake (the small
increase in counts shown can be attributed to
experimental error). At 3%(w/v) salts, approximately seawater concentration, the amino acid was
actively transported, though at a lower rate than at
15 or 25%(w/v) salt concentrations, which fall in
the range at which the microorganism can grow.
On the other hand, respiration was also detected at all the salt concentrations tested (Fig. 2).
In the medium with 0.5%(w/v) salts, the addition
of the substrate caused only a very small increase
of respiration rate, which cannot be considered
significant in this kind of experiments. However,
endogenous respiration detected in the flask
without added nutrients was noticeable, reaching a
total 02 consumption of 53.3/~1 after 8 h. This fact
@
I
r...------~[ ~ [
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/ / ':1-
2.3. Manometry
The cells were obtained as before and resuspended in 0.5, 3 or 15%(w/v) salts in equal
amounts to get a final suspension containing approx. 109 cells/ml. Oxygen uptake rates by such
suspensions were measured in Warburg constantv o l u m e m i c r o r e s p i r o m e t e r s at 41°C. The
organisms, together with enough salt solution of
the corresponding concentration to give a final
volume of 3 ml, were placed in the main compartment of the flask; 2 ml of 0.25%(w/v) yeast extract
(Difco) in the corresponding salt solution were
added in the side arm and 20%(w/v) aqueous
K O H solution (0.2 ml) in the centre well.
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~
e+++--+-°---~°~-~.
.._1
r
30
60
TIME, rain.
Fig. I. Effect of the salt concentration in the medium on the
transport of [14C]leucine: 0.5% ( w / v ) total salts (©
©)+
3% ( w / v ) (O
-O), 15% ( w / v ) (Z]
[]) and 25%
( w / v ) (m
m). Starting time was taken to be the moment
of addition of the radioactive amino acid. For details see
MATERIALS AND METHODS.
251
40
3(:
20
i-It,, ~
¢:1
10
2
4
TIME.
6
8
hours
Fig. 2. Effect of the salt concentration in the medium on the
respiratory rate of Halococcus sp. NCMB757: 0.5% (w/v) total
salts (O
O), 3% ( w / v ) (O
O) and 15% (w/v)
(U3
[]). Values of the endogenous respiration have been
subtracted.
seems to indicate that although no external substrate is metabolized, internal reservoirs of nutrients were being catabolized and respiration occurred even under these conditions. The absence
of external substrate consumption can be due to
the inability of the organisms to transport nutrients into the cell as in fact happens with the amino
acid studied.
Table 1
A m o u n t s of cell-associated K + retained by cells suspended
during one week in the total salt concentrations indicated
For the experimental method see M A T E R I A L S A N D METHODS
Total salts in
the suspending
medium %(w/v)
g K + / g protein
25
15
3
0.5
1.53
1.48
0.146
0.075
In Table 1 the amounts of cell-associated potassium retained in the cells after being suspended for
a week in the corresponding salt concentrations
are shown. It seems clear that K + was not retained
in the cells at low salt concentrations. The amounts
detected actually correspond to those expected,
considering that the intracellular concentration of
K + decreases concomitantly with the external concentration of salts, going down to very low values
at the seawater salt concentration.
The experiments described point out that the
state of the Halocoecus cells in seawater salt concentrations is very peculiar. The microorganism
seems to remain able to carry out many important
functions for the survival of the cell such as active
transport and respiration. However, growth does
not occur below concentrations of salts at least 3
times greater than that of seawater [3]. The question that arises then is why such high concentrations are required for growth. There are many
possible explanations from a physiological point
of view: the inhibition of just one of the numerous
processes implicated in growth, from D N A replication to cell wall synthesis, would be sufficient.
However, from an ecological point of view, it is to
be expected that for an extreme halophile, growth
would only be required and really productive in
hypersaline habitats where there is a high nutrient
concentration and less competition from other
bacteria [5].
The maintenance of the metabolic activities at
seawater salt concentrations described in this work
could play an important role in keeping Halococcus cells alive during long periods in seawater. It
is, however, very surprising how the enzymes required for these activities can function over such a
wide range of salt concentrations. In this respect
the enzymes of Halobacterium and even of many
moderate halophiles are thought to be much more
labile [ 1].
Finally, this is an interesting example in which
non-growing cells remain able to develop activities
oriented to the survival of the cell. This kind of
phenomenon is probably common in organisms
that live in very restricted habitats and may have
important ecological implications for their dispersion.
252
ACKNOWLEDGEMENTS
REFERENCES
Some of the experiments described were carried
out by F.R.-V. during a stay by in the Department
of Biology, University of Ottawa, Canada, and
supported by a grant from the National Sciences
and Engineering Research Council of Canada. We
thank Mr. F. Ant6n for the atomic absorption
determinations.
[l] Kushner, D.J. (1978) in Microbial Life in Extreme Environments (Kushner, D.J., Ed.) pp. 317-368, Academic Press.
London.
[2] Brown, A.D. and Cho, K.Y. (1970) J. Gen. Microbiol. 62,
267-270.
[3] Rodriguez-Valera, F., Ruiz-Berraquero, F. and RamosCormenzana, A. (1979) Appl. Environ. Microbiol. 38. 164165.
[4] Rodriguez-Valera, F., Ruiz-Berraquero, F. and RamosCormenzana, A. (1980) J. Gen. Microbiol. 119. 535-538.
[5] Larsen, H. (1980) in Hypersaline Brines and Evaporitic
Environments (Nissenbaum, A., Ed.), pp. 23 39, Elsevier,
Amsterdam.