Download The use of protein synthesis inhibitors in the estimation of the

FEMS MicrobiologyEcology73 (1990) 187-192
Published by Elsevier
187
FEMSEC 00249
The use of protein synthesis inhibitors in the estimation
of the contribution of halophilic archaebacteria
to bacterial activity in hypersaline environments
Aharon Oren
Division of Microbial and Molecular £cology. Institute of Life Sciences, The Hebrew Universityof Jerasalcm, Jerusalem, Israel
Received9 June 1989
Revision tefeived and accepted 2 October 1989
Key words: Halobacteriwn; Halophilic eubacteria; Saltern; Dead Sea; Chlorampheni¢6~; Anisomycin
1. S U M M A R Y
Antibiotics affecting protein synthesis were used
to differentiate between the activity of different
groups of orgazdsms (halophilic archaebacteria,
eubacteria and eukaryotes) in water samples from
hypersaline ecosystems. Ar, isomycin (inhibiting
both archaebacterial halophiles and eukaryotes)
can be used to quantitate the contribution of the
archaebacterial halophilcs to amino acid incorporation by the microbial community, when cyclohe~dmide (inhibiting eukaryotic protein synthesis,
but not affecting halobacteria) is used as a control. Both in sattem ponds at salinities above 300
g/1 and in Dead Sea surface water more than 95%
of the amino acid incorporation activity was
abolished by anisomycin, but not by cycloheximide. inhibition by anisomycin was well correlated with inhibition by low concentrations of bile
salts, which specifically affect bacteria of the
Haiobac,erium group. Chloramphenicol (an in-
Correspondence w: A. Omn, Division of Microbial and Molecular Ecology, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem919Pr¢,Israel.
hibitor of eubacterial prote~'..t synthesis) quantitatively inhibited amino acid incorporation in saltern
brines of r~lati~ely low salinity, but also caused
significant (28-42%) inhibit.ion at high salinities.
Erythromycin was also found valuable in the
estimation of activities of the different bacterial
groups.
2. I N T R O D U C T I O N
Hypersaline ecosystems are inhabited by different groups of m:.croorganisms. At salt concentrations approaching saturation red archaebacteria
(genera Halobacterium, Haloarcula, Haloferax,
Halococcus) are often present in numbers high
enough to impart a red color to the brines. In
addition, a variety of (moderately) halophilic or
halotolerant eubacteria are known to thrive at
high salt concentrations. Relatively little is known
of the ecology of the different bacterial groups
that develop at high salinities; studies based on
colony eotmts on agar plates showed that in
multi-pond salterns the ponds of low to intermediate salinity are dominated by halotolerant or
moderately halophilic eubacteria, while at the
0168.6496/90/$03.50 © 1990 Fod~afion of EuropeanMicrobiolo~calSodedes
188
highest salinities the archaebacterial types
dominate the community [1,2]. However, it should
be taken into account that plate count methods
are in general inefficient, and only a small percentage of the viable bacteria present may develop
into colonies; indeed, bacterial numbers reported
(2-4 x 104/ml) [1,2] are 2-3 orders of magnitude
lower than those observed microscopically in
salterns [3,4]. However, viable counts may be increased by using a mote efficient plating procedure that was recently developed [5].
The red halophiles (halobaeteria, halococci) belong to the archaebacterial kingdom, and as such
they differ greatly from the eubacteria in many
properties. These differences can be exploited to
obtain information on the relative importance of
the different groups in natural communities. Recently, specific lysis of halobacteria by low concentrations of bile salts [6] (a phenomenon based
on their special cell envelope structure) was used
to study the contribution of bacteria of the Halobacterium group to both bacterial community size
and activity in ecosystems of different salinities
[4]. In the present work I tested the possibility of
using the differences in sensitivity of the protein
synthesis machinery of archaebacteria, eubacteria
and eukaryotes to different antibiotics, in the study
of the ecology of hypersaline environments.
3. MATERIALS A N D
METHODS
3.1. Sampling sites
Samples were taken in May-July 1989 from
ponds of a solar saltern facility near Eliot, Israel
(sampling sites as described cartier) [4]. Dead Sea
surface water was sampled at the shore near Pin
Gedi.
3.2. Enumeration of bacteria
Bacterial densities in the brines examined were
estimated microscopically by means of a PetroffHauser counting chamber, using a microscope
equipped with phase optics. The conl,'.bution of
bacteria of the Halobacterium group to the
bacterial ¢xsmmunity was determined by comparing untreated samples with samples treated
with 0.025% sodium taurocholate (Sigma), as described previously [4].
3.3. Measurement of amino acid incorporation rates
The rate of amino acid uptake in brine samples
was determined by incubating samples (25 ml)
with shaking at 32°C in the presence of 25 /~1
[U-14C]protein hydrolysate (Amersham, 50 l~Ci/
ml, 57 mmol C/taCt) or 12.5/~1 L-[aSS]methionine
(Amersham, 16.2 mCi/mt, 244 mCi/mmol). The
basic procedure followed was as described earlier
[4]. Dead Sea brines were diluted to 80% (by
volume) with sterile distilled water. To some of
the flasks Na-taurocholate (50 /~g/ml), chloramphenicol (10 or 20/~g/ml), kanamycin, tetracycline, erythtomycin, anisomycin or cycioheximire (all at 40/~g/_ral) were added 30 rain prior to
the addition of the labelled amino acids. All antibiotics were obtained from Sigma Chemical Co.,
St. Louis, MO. At zero time and at ~uitable intervals 1-ml or 5-mi samples were withdrawn, filtered
on Millipore filters (0.45 pm pore size), the filters
were washed with 3 × 5 ml cold 10% trichloroacetic acid, and the radioactivity retained on thc~a
was determined. An~_ino acid incorporation rates
were calculated, assuming that the labelled amino
acids in the mixture had an average molecular
weight of 130 and a carbon content of 45%, and
that no significant amino acid pool was present in
the waters, which would have caused a decrease in
specific activity.
4. RESULTS
To estimate the contribution of the different
groups of microorganisms present in saltern ponds
of increasing so,airy, I examined the effect of
antibiotics affecting protein synthesis in different
~roups (archaebacteria, eubacteria, eukaryotes) on
the rates of incorporation of a mixture of r~dioactivel)" labelled L-amino acids (or L-mvthionine,
which gave similar results).
It was shown (Fig. 1) that cycloheximide, an
inhibitor specific for eukaryotic protein synthesis,
did not have a significant effect at any of the
salinities tested, and thus most activity must have
been due to bacterial action, and the contribution
189
7
0
1--<
90
7o
2:=.
_L
_
1
\\
u~
_.Ix
DISSC)LV~'D S A L T S
{9/[~
4oh
Fig. 1. Rates of amhlo acid incorpo~don by sa[tem samples of
different salinity, collected 14 May, ]989. and Eilat seawater,
at 320C, in the absence (o)and presence o[' different protein
synthesis inhibitors: ehlor~cmphcnicol (20 tt$/ml) ($): anisomycilt (40 #8/ml) (×); and cycloheximide (40 pg/ml)(A),
The lower parl of the figure shows the bacterial numbers
present as determined mic~mcopieally, in the absence (o) and
presence ( 0 ) of 0.02.5% Na-taurocholat¢.
of eukaryotic microorganisms can be considered
negligible.
To elucidate the relative contribution of
halophilic archaebacteria and eubacteria to amino
acid incorporation by the microbial communities
present, ardsom~cin or chloramphenicol was added to the assay systems. Anisomycin, a strong
inhibitor of halobacterial (and eukaryotic) ribosomes, did not have a significant effect in samples
of salinities below 150 g/l, but quantitatively inhibited amino acid incorporation at the highest
salinities (above 300 g/l). The inhibition by aniso*
mycin is not likely to be due to the activity of
eukaryotes, as it was shown above that cy:loheximide did not greatly affect the rates. It is thus
suggested that the activity of the microbial cornmunity in the high salinity saltem ponds can bc
attributed to bacteria of the Halobacterium group
only. This conclusion is supported by the finding
that those samples that were inhibited by ax~isomycin were also completely inhibited by low concentrations of taurocholate (Table 1), which was
shown earlier to abolish activity of tmlobacteria
[4]. The importance of the contribution of halobacteria to the bacterial community in ponds with
salt concentrations exceeding 300 $/1 was further
demonstrated by means of microscopic enumeration of bacteria before and after treatment with
taurocholate, which causes lysis of halobacteria
Table :1
Influence of different protein synthesis inhibitors and taurocboiate on amino acid ;ncorporation activity in saltern samples of
different salinity, and in Dead Sea surface water, as compared to water from a freshwater pond (botanical garden, the Hebrew
University, Jerusalem)
lnhibitors
Freshwater
None a
Ch]or alnphenico]
Chloramphenico!
Kanamycin
Erythromyein
Tetracycline
Cyeioheximide
Anisomyein
Na-taurocholate
100
7
6
5
5
16
98
ND
98
(10 p~'ffd)
(20 p g / m l )
(40 p s / m l )
(40 t t s / m l )
(dO/t g / n d )
(40 ~tg/ml)
(40 ~tg/nd)
(50 ~sJml)
Salterns (dissolved salts/t)
Dead Sea
131 g
228 g
381 g
100
ND
12
99
14
94
ND
73
06
100
ND
9
88
9
91
ND
t00
84
100
74
68
107
88
96
93
7
7
1O0
91
69
112
90
77
102
3
5
Incorporation rates ate expressed as percentages of Ihe rates oblained ~n ~h¢ ~,~n¢.~ of inhibitors (383, 43.4, E0.1 and 0.08 nmol
amino acids/t.h, r~¢Sl~Czive|y).ND, not determined.
190
(Fig. 1, lower part. compare Figs. 3 and 5 in an
earlier paper on the subject [4]).
Upon addition of chloramphenicol almost
quantitative inhibition of amino acid incorporation was obtained in the lower salinity range,
consonant with the dominance of eubacteria in
these waters, but even at the highest saIinities
some (28-42%) inhibition was observed. This inhibition is probably due to the action of chloramphenicol on halobacteria (see below), rather
than to the presence of active eubacteria, as shown
by the complete inhibition of activity by anisomycin in these samples. E~thromycin may give a
still better differentiation (Table 1). Kanamycin
and tetracycline, which should inhibit eubacterial
protein synthesis without affecting axcha~bacteria,
proved inactive in inhibiting amino acid incorporation in saitern ponds of low salinity (97-228
g/l).
Similar to the high-salinity saltern ponds, anisomycin completely inhibited amino acid uptake
in Dead Sea surface water samples, while chloramphenicol caused a 40~ inhibition, and
kanamycin, tetracycline, erythromycin and cycloheximide did not greatly affect uptake rates. Again
a correlation was found between inhibition by
anisomycin and low concentrations of bile salts
(Table 1), which were earlier reported to abolish
amino acid incorporation activity in Dead Sea
brines [7].
5. DISCUSSION
In the present study protein synthesis inhibitors
were used to obtain information on the relative
contribution of halophilic atchaebacteria of the
Halobacterium group and of halophilic eubacteria
to the bacterial activity in hypersaline ecosystems.
Few quantitative studies on the microbial ecology of hypersaline ecosystems such as salterns
have been reported, and most of these deal with
plate counts, followed by isolation and characterization of the bacteria present [1,2]. This approach
cannot but yield an incomplete picture, as the
percentage of bacteria that develop into colonies
on plates is generally low - colony numbers of up
to 4 × 104 should be compared to total bacterial
counts of 10"t/ml and higher (refs- 3, 4; this
study),and thus little understanding may be gained
on the nature of the dominant members of the
community. The use of bile salts, specifically affecting halobacteria when added in low concentrations, was shown earlier to add relevant information on the microbial ecology of hypersaline environments. A differential microscopic enumcxation procedure involving bile salts enables a more
reliable estimation, at least of the minimal numbers of halobacteria present. In addition, it was
shown that in brines from salterns at the highest
salinities amino acid incorporation activities were
abofished by low concentrations of bile salts, suggesting that only halobacteria are active at the
h i ~ e s t salt concentrations, and that bacterial types
insensitive to lysis by the bile salts used may not
contribute significantly to the overall activity [4].
The use of specific protein synthesis inhibitors
supplements the use of bile salts as inhibitors of
archaebacterial halophiles. Anisomycin is known
as a powerful inhibitor of eukaryo~ic protein
synthesis, binding at a single binding site on the
60S subunit of 80S ribosomes. In addition, it acts
on halophili¢ archaebacteria (Halobacterium
halobium, H. cutirubrum), both inhibiting growth
at low concentrations (2 pg/ml) [8,9], and in vitro
protein synthesis [10]. A good correlation was
found between inhibition of amino acid incorporation by bile salts and by anisomyein, both in
saltern ponds of different salinities, and in Dead
Sea brines. Theoretically, the use of bile salts does
not allow the differential measurement of
h',dophilic archaebacterial and ¢~bacterial activities, as it was shown that members of the genus
Halococcus are insensitive to bile salts. Practically
though, halococci cannot be expected to interfere
in the proposed differentiation between halophillc
archaebacterial and eubacterial activities by using
bile salts, as halococci have never been shown to
be of great quantitative importance in hypersaline
ecosystems. No reports were found on the sensitivity of halococci to anisomycin.
Chloramphenicol was used in this study to
abofish eubacterial protein synthesis. Neax-complete inhibition was observed in those environments in which eubacteria could be expected to be
the dominant component of the microbial community; however, in those brines dominated by
191
halophilie arehaebacteria (as evidenced by complete inlfibition of amino acid incorporation by
anisomyein or taurocholate) a sigrdficar,t extent of
inhibition (28-42%) was observed in the presence
of 20 #g/'ml chloramphenicol. This inhibition m a y
have been due to the action of chloramphenicol on
halophilic archaebacteria: while halobaeteria were
repor~,ed insensitDe to ehloramphenieol in agar
diffusion tests [11], inhibition was observed in
liquid cultures: H. haiobium grew at less than half
its optimal growth rate in the presence of 200
t~g/ml chloramphenicol [12l, while 3 1 / l g / m l was
reported to be the minimal inhibitory concentration to 1t. halobium and 1t. cutirubrum in a tube
dilution assay [9]. Methartogenic archaebacteria
are also inhibited by chlorampheni¢ol to a certain
extent [11,13,14]. Other cubacterial protein synthesis inhibitors such as tetracycline, erythromycin or
kanamycin were reported not to affect halobacteria [8,11t, and these did not inhibit amino acid
incorporation in Dead Sea water or salt ponds of
high salinity. Erythromycin enabled the differentiation of archaebacterial and eubactefial halophilic
bacteria. However, tetracycline and kanamyein did
not inhibit amino acid incorporation in those salt
ponds in which the activity was expected to be
primarily due to eubacteria. The reason for the
inefficiency of tetracycline and kanamycin in hypersaline eubacterial habitats was not examined
further.
Antibiotics have been used in the past in a
limi!ed number of ecological studies, e.g. to differentiate between activity of prokaryotes (eubacteria) and eukaryotes [15]. To my knowledge they
were never used before to quantitate the contribution of arehaebacteria to bacterial activities in
natural communities. Anisomyein turned out to be
an effective inhibitor of halobacterial activity,
which can be used in combination with other
protein synthesis inhibitors in ecological studies to
obtain information on the contribution of
halophilic arctiaebacteria to the total bacterial activity in hypersaline waters. The work 'described
demonstrates that the use of specific antibiotics
enables the exploitation of the profound differences between archaebacterial and eubacterial
halophiles, not only in physiology and biochemistry, but in ecological studies as well.
6. ACKNOWLEDGEMENTS
I thank the Israel Salt Co., Ltd. for allowing
access to the Eilat salt ponds, and the staff of the
lnteruniversity Institute of Eilat for laboratory
facilities and logistic support. This study was supported by grants from the Israeli Ministry of
Energy and Infrastructure and the Hebrew University of Jerusalem Mutual Fund.
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