Viruses, plasmids and other genetic elements of thermophilic and hyperthermophilic Archaea

12 Pages·2003·1.72 MB·English

YGOLOIBORCIM
SWEIVER
ELSEVIER SMEF ygoloiborciM sweiveR )6991 (81 225-236
Viruses, plasmids and other genetic elements of thermophilic and
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hyperthermophilic Archaea n
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Wolfram Zillig a,*, David Prangishvilli ,a Christa Schleper ,b Marieke Elferink ,a fro
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Ingelore Holz a, Sonja Albers ,a Davorin Janekovic ,a Dorothee G6tz c h
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a Max Planck-lnstitutfi~r Biochemie, 82152 Martinsried, Germany ://a
Marine b Sciences Institute, 'OisreuinU of California, Santa Barbara, CA 93106, ASU c
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c Unieersity of Waikato, Hamilton, New Zealand de
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Abstract .c
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We review and update the work on genetice lements, e.g., viruses and plasmids (exluding IS elementsa nd transposons) in /fem
the kingdom Crenarchaeota (Thermoproteales and Sulfolobales) and the orders Thermococcales and Thermoplasmales in sre
the kingdom Euryarchaeota of the archael domain, including unpublished data from our laboratory. The viruses of /a
Crenarchaeota represent four novel virus families. The Fuselloviridae represented by SSVI of S. shibatae and relatives in rtic
le
other Sulfolobus strains have the form of a tailed spindle. The envelope is highly hydrophobic. The DNA is double-stranded -a
b
and circular. Members of this group have also been found in Methanococcus and Haloarcula. The Lipothrixviridae (e.g., T s
TV 1 to 3) have the form of flexible filaments. They have a core containing linear double-stranded DNA and DNA-binding trac
proteins which is wrapped into a lipid membrane. The 'Bacilloviridae' (e.g., TTV4 and SIRV) are stiff rods lacking this t/1
8
membrane, but also featuring linear double-stranded DNA and DNA-binding proteins. Both virus types carry on both ends /2
-3
structures involved in the attachment to receptors. Both types are represented in Thermoproteus and Sulfolobus. The /2
2
droplet-formed novel Sulfolobus virus SNDV represents the 'Guttaviridae' containing circular double-stranded DNA. 5
/4
Though head and tail viruses distantly resembling T phages or lambdoid phages were seen electronmicroscopically in 7
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solfataric water samples, no such virus has so far been isolated. SSV1 is temperate, TTV1 causes lysis after induction, the 29
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other viruses found so far exist in cartier states. The hosts of all but TTVI survive virus production. We discuss the b
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implications of the nature of these viruses for understanding virus evolution. The plasmids found so far range in size from g
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4.5 kb to about 40 kb. Most of them occur in high copy number, probably due to the way of their detection. Most are e
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cryptic, pNOB8 is conjugative, the widespread pDLI0 alleviates in an unknown way autotrophic growth of its host t o
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Desulfurolobus by sulfur reduction. The plasmid pTIK4 appears to encode a killer function, pNOB8 has been used as a 0
9
vector for the transfer of the lac S (fl-galactosidase) gene into a mutant of S. solfataricus. A
p
Keywords: Archaea, ;cilihpomrehtrepyh Archaea, ;cilihpomreht Virus; ;atoeahcranerC ;selabolofluS ;selaccocomrehT :selamsalpomrehT ril 2
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226 W. Zillig et aL /FEMS Microbiology Reviews 18 (1996) 225-236
Contents
.1 Introduction ............................................................... 226
.2 Viruses of atoeahcranerC ...................................................... 722
2.1. Nature of the viruses ...................................................... 722
2.2. Virus host interactions ..................................................... 922
2.3. Plaque tests ............................................................ 230
2.4. Receptors ............................................................. 232
2.5. Assembly ............................................................. 232 Do
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.3 Origin and phylogeny of viruses ................................................... 232 loa
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.4 Plasmids ................................................................. 332 fro
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.5 'Sulfolobicins' . ............................................................ 432 ttp
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.6 Construction of transforming (cloning) vectors based on genetic elements .......................... 532 ://a
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Acknowledgements ............................................................ 532 m
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References ................................................................. 532 p
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1. Introduction Halobacterium halobium, pHH1 [6] have been stud- re/a
ied in molecular genetic detail. In the lowest branch rtic
Genetic elements, e.g., viruses and plasmids may of the Euryarchaeota, the Thermococcales, harbor- le-a
be considered 'fragments of life'. They constitute an ing the hyperthermophilic genera Thermococcus and bs
important fraction of biodiversity which is not con- Pyrococcus, several plasmids including pGT5 [7] trac
gruous to and appears to be largely independent of have been found recently, but no viruses are known t/1
8
that of their hosts and they have certainly partici- so far. In the second lowest branch of this kingdom, /2-3
pated in generating the variety of extant organisms the Thermoplasmales, viruses have not been de- /2
2
via recombination processes. Studying emergence scribed to date, but several plasmids were found 5/4
and scope of this diversity should help in understand- recently. The Pyrococcus plasmid pGT5 has been 79
2
ing origin and evolution of life. Moreover, such utilized as basis for the construction of a shuttle 97
elements supply small and thus comprehensible vector for .E coli, Sulfolobus and Pyrococcus by
g
genomes which have served in studying mechanisms (Aagaard and Garrett, personal communication). In u
e
and controls of DNA replication and gene expression Crenarchaeota, four viruses of Thermoproteus tenax st o
in Bacteria and Eucarya. Because of their capability (Thermoproteales) TTV1 to 3, representing the novel n 0
to be maintained and multiplied in their hosts, they virus family Lipothrixviridae, and TI'V4, represent- 9 A
p
hvaevcteo rs. been utilized for developing transformation ing' Bacilloviridae', another, not have been yet validated isolated virus (see f[3] amily, and [4]). the ril 20
1
The third domain of life, Archaea, has been Only TTVI has been investigated in more detail, 9
divided into two kingdoms, Crenarchaeota and Eur- including sequencing of 85% of its genome (see [8]).
yarchaeota [1]. A number of viruses and plasmids In the other order of the Crenarchaeota, Sulfolob-
have been described for Euryarchaeota, especially ales, the Sulfolobus virus SSVI, again representing
Halobacteriales (the viruses are listed in [2], earlier a novel virus family, the Fuselloviriodae, has been
reviews are [3] and [4]). Some of these, e.g., studied extensively, including complete sequencing
Halobacteriophage qbH [5] and a large plasmid of [9], mapping of transcripts and promoters, and analy-
W. Zillig et al. / FEMS Microbiology Re~'iews 18 (1996) 225-236 227
sis of lysogeny and of induction of virus production. E. coli phage P1 not only in structure and replication
Two novel Sulfolobus viruses, SIRV and DAFV of its DNA but also in the type of the lysogeny
have been found in isolates from Icelandic solfataras conferred to its host by a circular prophage. In view
[10]. SIRV another representative of the 'Bacillo- of the incompatibility of the transcription systems of
,L iridae' has been partially sequenced and character- Bacteria and Archaea [12], of the entirely different
ized (D. Prangishvilli and D. ,ztS~G unpublished work lifestyles of the hosts - life at low ionic strength for
from this laboratory). Our knowledge of DAFV of E. coli but at extreme salinity for Halobacterium -
Desulfurolobus ambivalens and of a novel Sul- and of the complexity of the viral genomes, stepwise
.[blobus virus from New Zealand, SNDV (W. Zillig, surmounting of the host range barriers between these D
o
D. ,ztS~G D. Janekovic and D. Langer, unpublished distant phyla during evolution appears highly im- w
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work from this laboratory) is, however, superficial. probable such that we feel forced to assume this loa
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Some l0 plasmids, most of them cryptic, but one virus type existed already before the separation of e
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conjugative, another one encoding a killer function the bacterial from the archaeal lineage, which is a fro
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and a third involved in autotrophic growth by sulfur very early event in the evolution of organisms [13,14]. h
reduction, have been found in Sulfolobales, the latter In contrast, to the euryarchaeotal viruses, those of ttp
s
in Desulfurolobus, the others in heterotrophic Sul- Crenarchaeota all represent previously unknown ://a
folobus strains from Iceland [10], Japan (Hokkaido virus families. The spindle-like virus SSVI of Sul- ca
d
1[ l]) and New Zealand (see below). folobus shibatae, solfataricus and islandicus repre- em
Here we present a short resum6 and an update of sents the Fuselloviridae which are characterized by ic.o
the state of the field, including unpublished material containing about 16 kb circular double-stranded up
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from our laboratory. DNA, a DNA-binding protein and a lipid membrane o
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made up of viral hydrophobic coat proteins and host /fe
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lipids. A short tail is visible but has not been anal- s
2. Viruses of Crenarchaeota ysed in detail. At least 7 viruses very similar in re/a
dimensions and shape and containing DNA of the rtic
.2 I. Nature of the t'iruses same size which cross-hybridized with that of SSVI, le-a
b
but was different in restriction patterns have been s
The viruses of extremely halophilic and found in novel Sulfolobus isolates from Icelandic trac
methanogenic Euryarchaeota are so far, with two solfataras (D. Prangishvilli and I. Holz, unpublished t/18
exceptions (see below), of the same head and tail work from this laboratory). A virus-like particle of /2-3
type as T phages and lambdoid phages of Bacteria Methanococcus t~oltae, VLP J2 15] [ and two viruses, /2
2
5
(see [2] and Table .)1 A striking example is the Hisl and His2 of Haloarcula hispanica (C. Bath and /4
7
temperate halobacteriophage q)H, which resembles M. Dyall Smith, personal communication) appear to 9
2
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Table 1 s
Viruses of crenarcaeota t on
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Designation Shape Size(length/width, rim) Type DNA size (kb) Sequence known Proteins Membrane Host Virus type 9
A
TTSVS VI 1 flex. ill. spindle 40010/04/060 lin. ccc ds 15.463 61 total 85% __>> 4 3 + + .S.T stoenialx . LFiupsotehlrlixotrJiirriiddaaee pril 2
0
TTV2 flex. ill. 1200/20 lin. ds 61 - > 1 + T. tenax. Lipothrixt'iridae 19
TTV3 flex. ill. 2500/30 lin. ds 27 - > 1 + T. tenax. Lipothrixciridae
DAFV flex. ill. 2200/27 lin. ds 56 - > 3 + D. arab. Lipothrixciridae
TTV4 stiff rod 500/30 lin. ds 17 - > 1 - T. tenax "Bacillot, iridae"
SIRV stiffrod 950/26 lin. ds 33 %51 _> 2 - S. isl. "Bacillociridae"
SNDV droplet 180/80 ccc 20 - > 61 ? S. spec. novel
822 .W Zillig et aL/ ReNews Microbiology FEMS 81 225-236 (1996)
be of the same type, which thus occurs in both pili of the host. They lack a membrane or hydropho-
archaeal kingdoms but not outside the archaeal do- bic coat. Early after discovery our 'isolate' of SIRV
main. turned out to be a mixture of variants which differed
A second novel family of viruses, the 'Bacillo- by restriction patterns of their DNAs but could not
viridae'(provisional name), has so far only been be distinguished by shape, size and SDS-PAGE pat-
found in Crenarchaeota, TTV4 in .T tenax (see [3], tern of viral proteins. Stable strains were obtained by
[4] and [16]) and SIRV (S. islandicus rod shaped cloning variants via single plaques. Though the nu-
virus) in S. islandicus [10]. These viruses are stiff cleotide sequences of corresponding genes of differ-
rods formed by a double helix consisting of linear ent variants (DNA-binding protein and dUTPase) D
o
double-stranded DNA and DNA-binding protein(s), showed up to 10% difference, the protein sequences w
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wound up to a tube terminally plugged and equipped were almost identical (D. Prangishvilli and D. Gt~tz, loa
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with tail fibers for adsorption, in the case of SIRV to unpublished work from this laboratory). e
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.giF .1 ekil-suriV selcitrap ni GEP setatipicerp of stnatanrepus of diuqil selpmas from cidnalecI ,sarataflos ni cases some dehcatta ot of cells
suetorpomrehT .ps ylevitageN .deniats
et w. Zillig al./ FEMS ygoloiborciM Reviews 81 225-236 (1996) 922
Viruses of a third novel family, the Lipothrixviri- large transcription units which are oriented back to
dae, again occurring in Crenarchaeota only, are back like pL and pR in E. coli phage .1 This
flexible filaments with a core of linear double- sequence is equipped with an unusual promoter,
stranded DNA plus DNA-binding proteins, wrapped which lacks the box A characteristic for archaeal
in a membrane consisting of host lipids and possibly standard promoters and seems to be barred by a host
hydrophobic viral proteins and equipped with tail repressor in the lysogenic state (T. Singer, diploma
structures apparently involved in adsorption to recep- thesis, L.M.U., Munich, Germany). Induction of virus
tors of the host. For three of these, TTV1 to 3, the production oc/I~K~curs via UV irradiation or mito-
host is T. tenax [2,3,17]. The fourth, DAFV, was mycin C or, very efficiently, via infection with the D
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found in a carrier state in a Desulfurolobus isolate virus SIRV, which is not multiplied under these w
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from Iceland [10]. conditions [10]. The lysogenic state is unique in that loa
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A fourth novel virus family is so far only repre- the components of the virus, except the DNA, are e
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sented by one large, droplet-like virus, SNDV, found present in the ceils, the coat proteins in regions of fro
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in a Sulfolobus isolate from New Zealand. The the host membrane and the DNA binding protein in h
pointed end of the droplet is densely covered by a the cytoplasm (W. Zillig, unpublished). What seems ttp
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beard of very thin, long fibers possibly involved in to be induced is DNA replication, possibly because ://a
the attachment to the host receptor (Fig. ;1 W. Zillig, the UV-inducible transcript, lind, acts as a primer. ca
d
D. Langer and D. Gtitz, unpublished data from our The newly synthesized DNA appears to be wrapped em
laboratory) The DNA is circular and has a size of 20 into its coat at virus membrane islands in the host ic.o
kb. A helical structure is recognizable on the surface membrane. No virus particles were ever seen in the up
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of the coat. The DNA is not split by many restriction cytoplasm of the host. o
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enzymes, indicating extensive chemical modifica- In the case of SIRV, linear viral DNA is found in /fe
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tion. the host when no virus is produced. This ill-defined s
These viruses were all found in cultures of novel carrier state may pass over into virus production in a re/a
Sulfolobus isolates. Virulent viruses, which might later growth phase of the culture of the virus carry- rtic
have been expected in liquid samples from hy- ing host or after infection of a virus-free host. After le-a
b
drothermal situations in solfataras, were so far not prolonged growth of a virus-carrying culture, curing s
encountered therein. Electron microscopy of concen- has also been observed. The controls involved in the trac
trates of such samples which had been obtained by virus host interaction are badly understood. The in- t/18
PEG precipitation and differential centrifugation, ex- fection of an SSV l lysogen with SIRV has, however, /2-3
hibited, however, various types of virus-like parti- the same consequences as UV irradiation and mito- /2
2
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cles, sometimes attached to putative Thermoproteus mycin C, which in E. coli both trigger the SOS /4
7
cells (Fig. 2). Some of these were of unusual head system. 9
2
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and tail types, others were spindle-like but differing TTV1 also occurs in a carder state in its host, .T 7
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in size from SSV1 in size, or rod-shaped, but differ- tenax, which in this case passes over into virus y
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ing from SIRV in dimensions. With the proper hosts, production when the carbon source has been almost u
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it might be possible to propagate such viruses. consumed [3,4]. As in the other systems, the cells t o
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2.2. Virus host interactions survive this productive phase. Nothing is yet known 09
on the relations of the other viruses to their hosts. A
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All these viruses were found associated with No lytic nor even truly virulent viruses have been ril 2
cloned host strains. In the case of SSV1, the mode of encountered so far, either because they did not sur- 0
1
coexistence is true lysogeny. The prophage is inte- vive outside of cells in the samples or because 9
grated in an arginyl tRNA gene by an integrase lysogeny or carrier states help to escape prolonged
whose gene is interrupted as the result of integration direct confrontation with the harsh solfataric envi-
[18,19]. A neighboring gene appears significantly ronment. This stress is illustrated by the observation,
similar to a consensus of exisionase genes. Virus that the DNA of SSV1 is rapidly inactivated by
production is switched on by the induction of the nicking when liberated virus remains exposed to the
transcription of a short DNA sequence between two optimal low pH, 3, in the culture medium (W.D.
230 W. Zillig et / al. FEMS Microbiology Reviews 18 (1996) 225-236
Reiter, diploma thesis, L.M.U. Munich Germany).
The pH of typical Sulfolobus habitats in solfataric
fields is as low as 1.7 to 2.5.
2.3. Plaque tests
Plaque tests, which are instrumental for molecular
genetic virus work are not available for the viruses
D
of the obligatory strict anaerobe .T tenax, which o
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requires elemental sulfur for growth and could not n
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yet be plated as a lawn. Moreover, the necessity to ad
e
handle this host in the absence of oxygen hinders the d
investigation of the interaction of virus and host. fro
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This was the major reason to concentrate our efforts h
on screening for genetic elements of aerobic, prefer- ttps
ably heterotrophic Sulfolobus strains. Virus-free het- ://a
c
a
erotrophic Sulfolobus species, e.g., S. solfataricus d
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and S. acidocaldarius yield good lawns when plated m
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in soft layers of about 0.2% gelrite, but not on .o
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surfaces of 0.6 to 0.8% gelrite gels [20]. p.c
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Good plaque tests have been established for SSVI m
[20], with S. solfataricus strain P1 as host and for /fe
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SIRV, with S. islandicus strain RN2H 1 as host [ .]01 sre
Because these viruses are not lytic, the plaques, /a
which are due to growth inhibition, are more or less rticle
turbid. In case of SSV1, they do not show sharp -a
b
s
edges. The quality of the plaques depends on the tra
growth state of the lawns which can be manipulated ct/1
by the seeding density. 8/2
From a single colony of a Sulfolobus strain from -3
/2
New Zealand, STH3/1, which carried the novel 2
5
virus SNDV, a cured daughter strain was obtained /47
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which upon infection produced this virus. No plaques 2
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were, however, recognized after infection of this host b
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with the virus, which is propagated in the late expo- g
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nential growth phase, where the lawn has almost e
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reached its final density (W. Zillig, unpublished). t o
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Fig. 2. Viruses of Thermoproteus tenax and Sulfolobus sp. Nega-
tively stained. (a) TTV1, (b) TTV2, (c) TTV3, (d) TTV4, (e)
DAFV, (f) DAFV within and getting out of cell of D. ambivalens,
(g) SIRV (courtesy of D. Typke and W. Baumeister), (h) particles
of SIRV spanning two pili of S. islandicus, (i) SNDV, (k) SSV1,
single particles and particles attached to membrane vesicle, )1(
same in the course of liberation from cell of S. shibatae, (m) o,s, urn
SNDV particles within cell of Sulfolobus sp.
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232 et w. Zillig .la / FEMS ygoloiborciM Reviews 81 225-236 (1996)
2.4. Receptors similarity to cellular genes and thus very probably
share a common origin with the latter. Another, more
Both TTV 1 and SIRV attach to pili of their hosts formal one is, that viruses by definition require hosts
(see Fig. 1), the former to the tips, the latter laterally. providing essential functions for propagation. Both
Both of these oblong viruses are on both ends arguments are not compelling. At least in the case of
equipped with structures allowing attachment to their rep proteins involved in RC type replication it ap-
receptors. As a consequence, SIRV can form 'string pears, that plasmid and viral genes branch off before
ladders' by forming bridges between two pili. the separation of Bacteria and Archaea [24]. Many
SSV1 attaches to the membranes of vesicles de- viral components do not find homologs in genomes D
o
void of an S layer. Its receptor appears, therefore, to of organisms, e.g., viral envelopes, RNA replicases w
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be situated in the membrane. Infection of a host with of RNA viruses and reverse transcriptases of retro- loa
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an intact S layer, e.g., S. solfataricus, thus poses a viruses etc. All components packed into the first e
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problem. It might occur at perturbations of S layer common ancestor of cellular life must have been fro
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structure which are responsible for the characteristic created in the course of prebiotic or chemical evolu- h
edged or angular appearance of Sulfolobus cells. tion and thus probably evolved there in correspon- ttp
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dence with each other and with many molecules and ://a
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2.5. Assembly systems which were not integrated into the first a
d
autonomous entity. Some of these could have formed em
The occurrence of complete virus particles in the partially autonomous assemblies, with small genomes ic.o
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cytoplasm indicates that Trv1 and DAFV are as- as essential components, depending on the metabolic p
.c
sembled within the cell. In contrast, massive extru- potential of the 'primordial soup' as 'superhost' in o
m
sion of SSV1 occurs in regions of the membrane much the same way as viruses on their hosts. After /fe
m
which are not or only loosely covered by an S layer the onset of cellular life such assemblies had sur- s
re
(Fig. 1) and no virus was ever seen within cells. We vived when they were able to replace the metabolic /a
have shown, that in SSV1 lysogens the highly hy- potential of the primordial soup (or whatever the rtic
le
drophobic viral coat proteins VP1 and VP3 are mem- scene of chemical evolution was) by that of a host. -a
b
brane proteins of the host while the DNA-binding Viral genes derived from cellular homologs could s
protein VP2 occurs in a particulate fraction of the have been added to viral genomes in the long period trac
cytoplasm (W. Zillig, unpublished). VP2 has been of interactive evolution of viruses and hosts. t/18
/2
shown to bind to circular DNA, especially viral As already discussed above, the similarities be- -3
DNA in a cooperative manner (W.D. Reiter, diploma tween the archaeal virus ~H and the bacterial /2
2
5
thesis, L.M.U., Munich, Germany). Taken together, phage P1 strongly indicate that this temperate phage /4
7
these observations indicate that VP2 is somehow type already existed before the separation of the 9
2
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stored inside the lysogenic cell, and forms a core Archaea from the Bacteria, which was the first 7
b
particle with viral DNA after DNA replication has documented lineage diversion in cellular evolution y
g
been induced. The latter then diffuses to islands of [13,14]. Further striking similarities between corre- ue
s
virus membrane in the cell membrane and is wrapped sponding features of an archaeal and a bacterial t o
n
there into its coat. Nothing is known yet on the virus host system are, (1) the close back to back 0
9
integration of tail structures into the virus particle arrangement of the promoters of the two large early A
p
nor on the assembly of the other viruses listed here. transcription units both in the archaeal virus SSV1 ril 2
(where they encode the early transcpts t5 and t6) and 0
1
the bacterial phage 1 (where they also code for the 9
3. Orion and phylogeny of viruses two major early transcripts [9]); (2) the position of
the UV inducible genes controlling virus production,
It is usually assumed that viruses are fragments of the tin d gene in SSV1 and the C1 gene in phage P1,
life which are composed of elements derived from between these promoters [9]; and (3) the site specific
organisms. This claim is based on several arguments. integration of the genomes of both viruses into the
One is, that many viral genes have, indeed, high host chromosome involving an integrase and an exci-
W. Zillig et al. / FEMS Microbiology Reviews 18 (1996) 225-236 332
sionase gene [19]. The resemblance of the latter Archaea and were thus possibly conserved only in
feature is even higher between SSVI and those that lineage or were in some way excluded from the
temperate bacteriophages and other bacterial genetic other domains. Lipothrixviridae and 'Bacilloviridae'
elements in which integration occurs in an arginyl have so far only been found in crenarchaeota and are
tRNA gene of the host [ .]81 therefore possibly younger than the deepest division
A particularly interesting hint about the early within the Archaea.
evolution of viruses is the finding that the circular Unfortunately, the fast evolution of viruses im-
genome of the Sulfolobus virus SSV1 consists of peding significant sequence comparison, and the ex-
two parts of almost equal size, one containing cys- cessive exchange of modules [21] between different D
o
teine codons in its ORFs, the other completely de- viruses prohibit construction of (possibly indepen- w
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void of these, the former encompassing the early dent) phylogenetic dendrograms for these genetic loa
d
transcription units for t5 and t6, the latter containing elements. e
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the gene cluster encoding some viral structural pro- fro
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teins and thus possibly other late functions too. h
These two functionally different halves of the SSV1 4. Plasmids ttp
s
genome, one using cysteine, the other not at all, ://a
c
appear to constitute 'modules' [21] of different ori- About a dozen plasmids have so far been identi- a
d
gin, the combination of which created the viral fied in Sulfolobus strains (Table 2). Due to the way em
genome. When that half which encodes similarities of isolation as cccDNA via ethidium bromide/CsCi ic.o
u
to functions of bacterial phages originated from an buoyant density centrifugation, most of these occur p
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ancestral situation already existing when Archaea in high copy number. Some are, however, not or o
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and Bacteria parted (see above) the other one, not barely seen in total DNA of their hosts or were only /fe
m
yet utilizing cysteine, could even have a more found when they were isolated via enrichment from s
re
primeval, possibly prebiotic origin. Because it con- larger amounts of total DNA, and thus appear to /a
tains the genes encoding structural proteins of the occur in low copy number. rtic
le
virus, e.g., the viral envelope, which so far does not Most of these plasmids are cryptic, i.e., no func- -a
b
find homologies in organismic structures, viruses tions except maintenance have yet been uncovered. s
might indeed have existed before organismic life However, a plasmid of Desulfurolobus ambivalens, trac
started. pDLI0, has been found in 20 of 22 independent t/18
On the other hand, representatives of the isolates of this relative of Sulfolobus, which thrives /2-3
Fuselloviridae, e.g., SSVI, have only been found in autotrophically, alternatively by the oxidation or by /22
5
/4
7
9
Table 2 2
9
Plasmids of sulfolobales 7 b
y
Designation Host DNA size Copy number Function g
u
e
pRNI S. islandicus 5.5 20 crypto s
pRN2 S. islandicus 6.9 53 crypto t o
n
pilE7 S. islandicus 7.5 51 crypto 0
9
pSTH4 S. spec. (N.Z) 4.7 high crypto A
pTpWAHUI4I S. S. ssppeecc.. (N.Z) (N.Z) 6.2 6.8 high low crypto crypto pril 2
0
pWHI2 S. spec. (N.Z) 4.51 high crypto 19
pTIK4 S. spec. (N.Z) 3.41 high Sulfolobicin?
pSTH3 S. spec. (N.Z) 35.0 high crypto
pNOB8 S. spec. (Jap.) 45 high, varies conjugative
pDL10 D. ambivalens(in most isolates) 7 high (to 50?) H2S-autotrophy?
pKAW 1 P. oshimae 8.3 high crypto
pKAW2 P. oshimae 8.8 high crypto
pTAI T. acidophilium 2.51 ~ 01 crypto
234 W. Zillig et / al. FEMS Microbiology Reviews 18 (1996) 225-236
the reduction of sulfur. Its copy number is strongly Pyrococcus element pGT5 [24] have been sequenced
enhanced during growth by sulfur reduction though so far. Each of these sequences contains two ORFs
the plasmid is not absolutely required for this mode one of which is large and has significant similarity
of existence. The widespread occurrence of this ele- with per genes involved in the maintenance, e.g.,
ment indicates the existence of a spreading mecha- replication of such elements in the cell via a rolling
nism while the linkage between copy number and circle mechanism. This notion is supported by the
mode of energy metabolism points at some func- occurrence of origin motifs known to be used in RC
tional, though not stringent, involvement, in growth replication [24].
by sulfur reduction [22]. Two plasmids, one of 8.3, the other of 8.8 kb, D
o
Another plasmid, pTIK4 from New Zealand, en- have been found in two different strains of the w
n
ables its host, to outgrow or kill competing Sul- moderately thermophilic hyperacidophile Pi- loa
d
folobus strains without producing a diffusible toxic crophilus oshimae, a relative of Thermoplasma aci- e
d
agent, possibly by cell contact. Colonies of this dophilum from a Japanese (Hokkaido) solfatara [25]. fro
m
isolate spread fast on gelrite plates by swarming (W. They are both contained in the strain DSM 9789 kept h
Zillig, unpublished). by the Deutsche Sammlung von Mikroorganismen, ttp
s
The conjugative plasmid pNOB8 from Japan the larger one as major, the smaller as minor compo- ://a
(Hokkaido) is efficiently transferred from donor to nent. The two plasmids share yet unmapped cross- ca
d
recipient cells via cell to cell contacts [11]. The hybridizing sequences. A plasmid of 15.2 kb has em
transfer mechanism is unknown. After transfer, the recently been found in Thermoplasma acidophilum ic
.o
copy number (per host chromosome) is high, but [26]. up
.c
colonies obtained by plating on gelrite yield liquid o
m
cultures with low copy number. The mechanism of /fe
m
copy number control is not understood but the low 5. 'Sulfolobicins' s
copy number state can also be approached by serial re/a
transfer of the transcipient. We have been able to About 30 of some 300 glycerol conserves of rtic
obtain Sulfolobus strains containing both the cryptic novel Sulfolobus strains from Iceland which had le-a
plasmid pilE7 and the conjugative plasmid pNOB8 been cloned from single colonies formed sharp-edged bs
by electroporating a recipient already carrying the halos upon dotting on lawns of selected virus-free trac
former plasmid with the latter, followed by allowing hosts (D. Prangishvilli, unpublished results from our t/1
8
conjugative spread of pNOB8 in the culture contain- laboratory). These halos resulted from complete inhi- /2-3
ing the primary transcipients. Immediately after bition of the growth of the lawn around the dot. No /2
2
5
spreading, the copy numbers of both plasmids were virus particle nor infectious agent responsible for this /4
7
high. In strains obtained from single colonies on phenomenon could be discovered. Instead, a dif- 9
2
gelrite plates, they were so low that the plasmids fusible macromolecular, apparently non proteina- 97
b
could only be recognized by Southern hybridization ceous cytotoxic agent was concentrated from the y
g
and one or the other had been lost in some of these cell-free supernatant of the culture of one of the u
e
s
strains. The replication of both plasmids appears thus strains. The toxin producing strain was resistant t o
to be controlled in the same way. When a donor against its own product. The chemical nature of the n 0
9
containing both pNOB8 and the small cryptic plas- agent and the mode of its action are unknown. But it A
fmli-dg alactosidase pilE7 was matneedg ative with recipient containing .S solfataricus PHI neither as a dsuegfgeesntsse in itself the that competition these agents with could other be means Sulfolobus of pril 20
1
plasmid, pNOB8 spread in the culture but transfer of strains. All isolates from an enrichment culture giv- 9
pilE7 was not observed. This indicates specific ing rise to one such strain indeed shared the capacity
transfer of pNOB8 (S. Albers and I. Holz, unpub- to excrete such toxins. Since toxin production is a
lished work from our laboratory) rather than trans- unique property - the high frequency of observing it
port through a cytoplasmatic bridge as in a mating results probably from selection during enrichment -
mode observed with Haloferax volcanii [23]. genes for toxin synthesis and insensitivity towards
Only two of all these plasmids, pRN1 [23] and the toxin action might be encoded on plasmids.