FEMS MicrobiologyLetters 55 (1988) 235-240
Published by Elsevier
235
FEM 03309
Distribution of reverse gyrase in representative species
of eubacteria and archaebacteria
R . G . Collin, H . W . M o r g a n , D . R . Musgrave, R.M. D a n i e l
Department of Biological Sciences, University of Waikato, Hamilton, New Zealand
Received 30 September1987
Revision receivedand accepted 6 June 1988
Key words: Archaebacterium; Reverse gyrase; Topoisomerase; Thermoacidophiles, D N A gyrase
1. S U M M A R Y
Reverse gyrase is a topoisomerase which positively supercoils closed circular plasmid DNA.
Reverse gyrase activity is restricted to the thermoacidophilic group of archaebacteria. Thermophilic
methanogens and eubacteria and all mesophilic
organisms screened had no reverse gyrase activity.
The result supports the deep phylogenetic divergence in archaebacterial evolution.
2. I N T R O D U C T I O N
A growing list of fundamental cellular properties supports the concept that archaebacteria are
phylogenetically distinct from the eubacteria and
eukaryotes. Organisms assigned to the archaebacterial kingdom show a deep evolutionary divergence within the kingdom which reflects their
habitat and physiology. The methanogens are
strictly anaerobic producers of methane from hydrogen and carbon dioxide, the halophiles are
aerobes with a requirement for high salinities and
Correspondence to: R.G. Collin, Department of Biological Sciences, Universityof Waikato, Hamilton, New Zealand.
the thermoacidophiles include aerobic and
anaerobic species all of which grow at high temperatures and either oxidise or reduce elemental
sulphur [1]. Largely on the basis of 16S rRNA
sequence homology the archaebacteria were divided into two sub-branches, the methanogens
plus halophiles and the thermoacidophiles [2]. The
wall-less thermoacidophile, Thermoplasma, is notable for having a closer affinity with the
m e t h a n o g e n sub-branch than the thermoacidophilic sub-branch. More recently, comparisons of complete 16S RNA sequences [3] and
D N A / r R N A hybridization velocities [4] suggest
that species of Thermococcus and Pyrococcus, both
of which are thermoacidophiles, may constitute a
third branch of the kingdom [3].
From evidence of differences in ribosome morphology, Lake et al. [5] consider the primary
evolutionary split between thermoacidophiles and
other archaebacteria to warrant the removal of the
former to a separate k i n g d o m - - t h e Eocytes.
Eocytes were defined as a kingdom with a close
relationship to eukaryotes and an extension of this
approach suggested a common origin of photosynthesis in eubacteria and halobacteria [6]. Both
proposals have been criticised for the lack of
supporting evidence [7,8]. Whether the presently
accepted archaebacterial kingdom should be split
0378-1097/88/$03.50 © 1988 Federation of European MicrobiologicalSocieties
236
or remain as a valid taxon, albeit a paraphyletic
one, may remain a semantic question unless more
evidence is obtained on fundamental cellular
properties. Much of the present evidence pertains
to ribosome morphology and ribosome components. Apart from the presence or absence of
introns [3] relatively little attention has been paid
to the genome itself.
Recently, an unusual topoisomerase (reverse
gyrase) from Sulfolobus acidocaldarius, which positively supercoils D N A in vitro, was described [9].
The enzyme was unique for a type I topoisomerase in that it required ATP and could convert negatively supercoiled DNA step wise to a
positively supercoiled form [10]. Reverse gyrase
activity has not been reported for any other
organism. An attempt to detect reverse gyrase
using an antiserum raised against the Sulfolobus
enzyme gave negative results when tested against
crude extracts of E. coli, Thermus thermophilus,
Halobacterium halobium, yeast and Drosophila
melanogaster [11]. No other sulphur-dependent or
thermophilic archaebacteria were screened. DNA
topoisomerases are a ubiquitous class of enzymes
that participate in many vital cellular reactions
involving DNA. This central role is indicated by
the sensitivity of archaebacteria, eubacteria and
eukaryotic cells to drugs which either inhibit or
modify the action of these enzymes [12].
While the in vivo role of reverse gyrase is
unknown, it is significant that the genome of the
S. acidocaldarius virus SSVI exists in a positively
supercoiled state [13]. Neither the organisation nor
the topological state of the chromosomal DNA in
Sulfolobus is known; however the predominance
of reverse gyrase activity suggests that a positively
supercoiled genome (or at least positively supercoiled domains) may exist. This has suggested a
number of possible roles for reverse gyrase. Firstly,
reverse gyrase may be related to the thermostability of S. acidocaldarius DNA, preventing denaturation at high temperatures by introducing
positive superhelical constraints into the genome
[9]. A second possible role is that reverse gyrase
counteracts the effects of other topoisomerases.
Thirdly the enzyme could transform cruciform
structures or domains of Z-DNA back to the
regular B-DNA. The transition of such structures
in chromosomes may be important as initiation
signals for D N A replication and recombination,
or gene expression [14]. The latter two suggestions
imply fundamental differences in cellular regulation between S. acidocaldarius and other
organisms. The distribution of reverse gyrase activity in other phyla may therefore be of use
phylogenetically. This paper reports the distribution of reverse gyrase activity in representative
mesophilic and thermophilic eubacteria and
archaebacteria.
3. MATERIALS A N D M E T H O D S
3.1. Source of Cultures
Cultures were obtained from either the American Type Culture Collection (A.T.C.C.), the German Collection of Microorganisms (D.S.M.) or
isolated locally. Recommended media were used
for all cultures except isolates of Thermoproteus
and Desulfurococcus which were grown on the
following medium prepared anaerobically (g-1-1
distilled water): (NH4)2SO4, 1.3; CaC12-2H20,
0.074; MgSO 4 • 7H20, 0.28; K H 2 P O 4, 0.28;
L-cystine 0.5; N a 2 S . 9H20, 0.1; yeast extract, 0.3;
trypticase peptone, 2.0; resazurin (0.1%), 1 ml;
Nitsch's trace elements, 1 ml; p H was adjusted to
6.0 at 25 ° C.
Cells were grown to stationary phase at the
recommended optimum temperature and harvested
by centrifugation at 13 000 x g for 20 min. Pellets
were resuspended (10 x v / w ) in lysis buffer (50
mM Tris-HC1 pH 7.5; 0.6 M NaCI; 1 mM
spermidine; 1 mM phenylmethyl-sulfonyl fluoride; 5 mM mercaptoethanol). Cells were lysed by
sonication, centrifuged at 13000 x g for 15 min
and the supernatant stored in 20% glycerol at
- 2 0 ° C until required for assay.
For reverse gyrase assay 1/~1 of cell lysate was
incubated with 0.5 /~g negatively supercoiled
pBR322 plasmid DNA for 10 min in assay buffer.
Assay buffer contained 50 m M Tris-HC1 p H 7.5;
0.2 M NaC1; 1 mM spermidine; 10 mM MgCI2; 1
mM dithiothreitol; 1 mM ATP. Assays were carried out at 75 ° C for all thermophilic cultures and
at the optimum growth temperature for other cultures. The reaction was stopped by adding 1 / 5
237
volume of a solution containing 5% sodium dodecyl sulfate, 50 mM EDTA; 50% glycerol and 0.05%
bromophenol blue. Positive and negative supercoils were separated using the technique of two
dimensional gel electrophoresis on 1% agarose gels.
After running in the first dimension gels were left
for one hour in the electrophoresis buffer containing 0.005 # g - m l - 1 ethidium bromide before running the second dimension.
For Halobacterium and Halococcus cultures a
range of NaC1 and KCI concentrations from 1 to 5
M were substituted for the NaC1 c o m p o n e n t of
the lysis and assay buffers.
4. RESULTS AND DISCUSSION
Under the conditions of assay reverse gyrase
activity was detected by the formation of posi-
Table 1
Species
Pseudomonas fluorescens
Escherichia coli
Clostridium thermoaceticum
Clostridium thermohydrosulfuricum
Thermobacteroides acetoethylicus
Bacillus stearothermophilus
Bacillus acidocaldarius
Bacillus caldovelox
Bacillus caldolyticus
Bacillus caldotenax
Thermus thermophilus
Thermus s p
Thermotoga s p
Halobacterium halobium *
Halobacterium saccharovorum *
Methanosarcina barkeri *
Strain
Optimum
Reverse
number
growth
gyrase
temperature
present
ATCC
13525
30 o C
-
HB
101
D S M 521
37 ° C
55 o C
-
DSM 570
60 ° C
-
ATCC 33265
DSM 22
65 ° C
55 o C
-
ATCC
27009
-
60 ° C
-
D S M 411
70 ° C
-
DSM 405
70 o C
-
DSM 406
70 ° C
-
ATCC
27009
60 ° C
-
ATCC
31674
75 ° C
-
Strain FjSS3-B.1
80 o C
-
DSM 670
ATCC 29252
37 ° C
37 o C
-
unknown
37 ° C
-
strain
Methanobacterium thermoautotrophicum
Methanococcus thermolithotrophicus *
Thermoplasma acidophilum *
Sulfolobus acidocaldarius *
Sulfolobus acidocaldarius *
Sulfolobus s p *
Sulfolobus solfataricus *
Desulfurococcus mobilis *
Desulfurococcus mucosus *
Desulfurococcus s p *
*
ATCC
65 ° C
-
DSM 2095
29096
65 o C
-
ATCC
25905
55 ° C
-
FERM
P.7137
75 ° C
+
ATCC 33909
75 o C
+
Strain pool 70
75 ° C
+
DSM
+
1616
80 ° C
DSM 2161
85 o C
+
DSM 2162
85 ° C
+
Strain TOK
85 ° C
+
85 o C
+
85 ° C
+
12 S.1
Desulfurococcus s p *
Strain RT
5 9 S.1
Desulfurococcus s p *
Strain KET
55 S.1
Thermoproteus lenax *
Thermoproteus tenax *
Thermoproteus tenax *
DSM 2078
85 ° C
+
Strain H 3
Strain TOK
12 S.2
85 ° C
85 ° C
+
+
Thermococcus celer *
DSM 2476
85 ° C
+
75°C
+
Isolate AN1 *
* Denotes
an archaebacterium.
238
Fig. 1. Two-dimensional gel electrophoresis of pBR322 DNA
exposed to cell extract of Desulfurococcus mobilis DSM 2161.
Negatively supercoiled topoisomers are on the left hand side of
the arch, positively supercoiled topoisomers on the right. Relaxed circular DNA is at the top of the arch; the bright spot to
the left of open circle topoisomers represents nicked circles.
tively supercoiled topoisomers f r o m negatively supercoiled pBR322. The typical ' l a d d e r ' of topoisomers formed b y the enzyme is illustrated in Fig.
1. With the use of crude cell extracts the result
obtained reflects the balance of competing enzyme
activities and must be interpreted with caution.
The use of a negatively supercoiled substrate
means that true gyrase activity would not be detected. F o r some thermophilic eubacteria only
negatively supercoiled topoisomers were detected
(the left h a n d side only of the ladder in Fig. 1)
indicating that the negatively supercoiled substrate was being relaxed. We c a n n o t rule out the
possibility that reverse gyrase activity was present
in these cell extracts but was masked by a more
active gyrase function. Concomitantly, where reverse gyrase activity was detected this represents
the nett result of competing supercoiling and
negative supercoiling functions in the extract.
The distribution of reverse gyrase in representative archaebacteria and eubacteria is summarised
in Table 1. Reverse gyrase is specific to the thermoacidophilic archaebacteria. Eubacterial thermo-
philes, including a Thermotoga strain which grows
in the same temperature range as archaebacteria,
[15] showed no positive supercoiling activity. Reverse gyrase activity was also absent from all
methanogens and halophiles screened, including
the thermophilic species. In agreement with recent
proposals [3] Thermoplasma, which although
phylogenetically unique shows a closer relationship with the methanogens, did not show reverse
gyrase activity.
The position of an isolate from N e w Zealand
(ANI), Thermococcus celer and species of Pyrocococcus within the thermoacidophilic b r a n c h of
the archaebacterial phylogeny has recently been
re-evaluated [3,4] with the proposal that a third
division of the archaebacterial k i n g d o m for these
organisms would be valid based on the phylogenetic depth of the group. If such is the case,
reverse gyrase activity was p r e s u m a b l y present
early in the evolution of the thermoacidophiles
but after the separation of the m e t h a n o g e n s and
halophiles. Isolate A N I has a temperature optim u m more typical of eubacterial thermophiles
( 7 5 ° C ) reinforcing the contention that reverse
gyrase is not a requirement for growth at ultra-high
temperatures. The distribution o f reverse gyrase is
restricted to the Eocytes, as p r o p o s e d by Lake et
al. [5]. However, a single t a x o n o m i c character
cannot be used to substantiate or refute such a
proposal and provides no evidence of phylogenetic
depth. If the D N A of these organisms is in a
positively supercoiled configuration in vivo then
more fundamental differences in gene expression
and genetic exchange might be expected.
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