Microbiology Journal Club
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Sept 13, 2005 - Jim Brown
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The papers for today are:
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A monogram: Huber H, Hohn MJ, Rachel R, Fuchs T, Wimmer VC, Stetter KO.
2002. A new phylum of Archaea represented by a nanosized hyperthermophilic
symbiont. Nature 417:63-7.
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A genome sequence: Waters E, Hohn MJ, Ahel I, Graham DE, Adams MD,
Barnstead M, Beeson KY, Bibbs L, Bolanos R, Keller M, Kretz K, Lin X, Mathur E,
Ni J, Podar M, Richardson T, Sutton GG, Simon M, Soll D, Stetter KO, Short JM,
Noordewier M. 2003. The genome of Nanoarchaeum equitans: insights into early
evolution and derived parasitism. Proc Natl Acad Sci U S A. 10:12984-8.
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Nanoarchaeum equitans
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Obligate ectoparasite of Ignicoccus sp. - both are Archaea
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Hyperthermophilic
0.4um diameter cocci - similar in size to Ultramicrobacterium
and Mimivirus
Potentially a new archaeal Kingdom
Very small genome - 490,885bp circle
Disorganized genes - little gene clustering and lots of split
genes, including both protein and RNAs
The University of Regensburg
Karl
Stetter
In situ,
Yellowstone National
Park, USA
Harald Huber lab
Ignicoccus
Autotrophic - Sulphur-reducer
Thermophilic - 90C optimum
Huber H, Hohn MJ, Rachel R, Fuchs T, Wimmer VC, Stetter KO. 2002. A new phylum of
Archaea represented by a nanosized hyperthermophilic symbiont. Nature 417:63-7.
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First thought to be cytoplasmic blebs or buds of Ignicoccus
But...
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They stain with DAPI
“universal” rDNA PCRs only yeild the Ignicoccus rRNA sequence
They don’t light up with Ignicoccus-specific rRNA probes.
Isolated blebs won’t grow
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be colonized by the tiny cocci. The final density of both the tiny cocci
and the Ignicoccus cells was about 3 £ 107 cells ml21 resulting in
about two tiny cocci per Ignicoccus cell on average. The purified
coculture was used in all further investigations. Cloning of single
Ignicoccus cells gave rise to cultures which never contained tiny
cocci.
By electron microscopy, a close attachment of the tiny cocci to the
surface of the Ignicoccus cells became evident (Fig. 1a, b, c). The tiny
But DNA isolated from cocci consistently exhibited a cell diameter of about 400 nm. In
contrast to Ignicoccus, they were covered by a regular surface layer
isolated (”filter sterilized”)
(S-layer) with sixfold symmetry and a lattice constant of 15 nm
(Fig. 1a). Ultrathin sections showed the presence of cytoplasmic
blebs does light up with
universal rDNA probes in
Southerns, and is different
that Ignicoccus rDNA
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Table 1 Se
Designation
.....................
8aF12
8mcF
9bF12
So, blebs seem to contain
DNA and rDNA distinct
from Ignicoccus, but the
rRNA won’t hybridize in
situ with “universal”
probes.
217kF29
Figure 2 Southern blot analysis of DNA from Ignicoccus sp. and the ‘N. equitans’–
Ignicoccus sp. coculture, treated with restriction enzymes. S, length standard (DNA
relative molecular mass marker III, Roche). Lane 1 and 3, Ignicoccus DNA. Lane 2 and 4,
DNA of the coculture. Lane 1 and 2, digested with EcoRI. Lane 3 and 4, digested with
HindIII.
64
helices n
identitie
(0.69–0.
range as
0.83). Th
the basis
(the dwa
equitans
In ss r
519uF12
519mcF
1114aR
1114mcR
1406uR12
1406mcR
1513uR12
1513mcR
EURY498R
511mcR
CREN499R
515mcR
ARCH915R
934mcR
.....................
C, Crenarc
Bacteria. Ba
© 2002 Macmillan Magaz
‘N. equitans’ represents an isolated, very deeply branching lineage.
However, because of its unique ss rRNA, a large variation of its
mosome) suggests that the N. equitans genome is evolutionarily
branching point, challenged by insignificant bootstrap values below
50%, was observed (not shown). Therefore, the determination of
stable compared with many bacterial parasites.
the accurate branching position of the ‘Nanoarchaeota’ must await
Owing to their great divergences in ss rRNA—in contrast to
the Ignicoccus host—cells of the ‘Nanoarchaeota’ did not stain
by fluorescence in situ hybridization using ss rRNA-targeted oligonucleotide probes directed against Crenarchaeota and Euryarchaeota (for example, EURY498R15, CREN499R15 and ARCH915R16;
MICROBIOLOGY
motifs for
f NEQ528
alanyl–tRNA
ere adapted
The enzymes
1 N-terminal
gles), and N.
Fig. 3. Phylogenetic position of N. equitans within the Archaea. The tree was
determined by the maximum likelihood method, based on 35 concatenated
ribosomal protein sequences. Numbers indicate percentage of bootstrap
resamplings. The scale bar corresponds to 10 estimated substitutions per 100
amino acid positions.
Figure 3 Secondary structure model for the ss rRNA of ‘N. equitans’. Highlighted positions indicate characteristic archaeal secondary structures11. Numbers correspond to the helix
numbers. The model structure was determined using RnaViz28.
NATURE | VOL 417 | 2 MAY 2002 | www.nature.com
PNAS ! October 28, 2003 ! vol. 100 ! no. 22 ! 12987
© 2002 Macmillan Magazines Ltd
ssu-rRNA shows that the “blebs” are actually an entirely different organism; an archaeon,
but novel and distinct from Euryarchaea, Crenarchaea, and Korarchaea - a new Kingdom!
65
Nanoarchaeum equitans
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“the dwarf archaeon riding the fire
sphere”
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Grows only attached to Ignicoccus, not
in extracts or separated in coculture
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Parasitic - slows the growth of
Ignicoccus at high MOI; the only
known archaeal parasite
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ca. 400nm coccus, ca. 500kbp genome;
both in the size range of the smallest
genomes or cells, or the largest
viruses or viral genomes
Waters E, Hohn MJ, Ahel I, Graham DE, Adams MD, Barnstead M, Beeson KY, Bibbs L,
Bolanos R, Keller M, Kretz K, Lin X, Mathur E, Ni J, Podar M, Richardson T, Sutton GG,
Simon M, Soll D, Stetter KO, Short JM, Noordewier M. 2003. The genome of
Nanoarchaeum equitans: insights into early evolution and derived parasitism. Proc Natl
Acad Sci U S A. 10:12984-8.
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The N. equitans genome:
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A single 490,885 bp circle (the smallest genome of any cellular organism known)
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Few or no pseudogenes - no longer undergoing reductive evolution
31.6% G+C - thermophiles do not have high GC contents!
552 protein-coding genes, unlinked single-copy rRNA genes, 38 tRNA genes,
snoRNA genes, etc, cover 95% of the genome. Very high gene density!
2/3rds of protein-encoding genes can be assigned
Little gene clustering (operons). Even ribosomal proteins and tRNA genes are
encoded separately.
The N. equitans genome doesn’t have:
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No genes for chemoautotrophic metabolism
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No enzymes for gylocolysis, gluconeogenesis, pentose
phosphate pathway, TCA cycle
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No SRP or RNase P subunits
Few enzymes for synthesis of aa, nucleotides, cofactors, even
lipids!
3 tRNA genes are absent; His, Glu Trp (but not really.....)
SAM synthase (but has enzymes that require SAM)
The N. equitans genome does have:
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Minimal ATPase - for generating proton gradient? Is it an
energy parasite?
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Some transporters, but not enough for everything it needs
Flagella biosynthetic apparatus (but they’re non-motile!)
HSPs and proteosomes
Inteins and typical tRNA introns
A full set of replication, repair, transcription, translation, and
cell cycle machinery
Split genes
Table 1. Split noncontiguous genes in N. equitans
Gene
Large helicase-related protein
Topoisomerase I
DNA polymerase I*
Archaeosine tRNA–guanine
transglycosylase†
RNA polymerase subunit B‡
Glu–tRNAGln amidotransferase (gatE)
Reverse gyrase§
Hypothetical RNA-binding protein
Hypothetical protein
Alanyl–tRNA synthetase
CDS encoding CDS encoding
N-terminal part C-terminal part
NEQ003
NEQ045
NEQ068
NEQ124
NEQ409
NEQ324
NEQ528
NEQ305
NEQ173
NEQ245
NEQ434
NEQ438
NEQ495
NEQ547
NEQ156
NEQ396
NEQ318
NEQ506
NEQ096
NEQ211
*Also split in Methanothermobacter thermautotrophicus.
†Also split in Methanopyrus kandleri, the Methanosarcinales, A. fulgidus, the
extreme halophiles and crenarchaea.
‡Also split in methanogens, A. fulgidus, and extreme halophiles.
§Also split in Methanopyrus kandleri (different site).
equitans, the split sites for most of these genes lie between
functional domains of the encoded proteins; thus, it seems likely
that the two separated genes are expressed to form subunits of
a functional enzyme. The genes for two subunits of alanyl–tRNA
encodes the F and G motifs. Toge
although the two genes are separ
mosome. We predict that the two p
are expressed separately and then
reassembled intein has been excis
nism, as observed in the DnaE p
(42). Topoisomerase I and revers
contain inteins in some archaea; h
were detected in these split genes
The N. equitans reverse gyrase i
encoding a helicase (NEQ434) and
domain. Reverse gyrase appears t
helicase and a topoisomerase do
supercoil formation in DNA (43
enzyme is present only in hyperth
that hyperthermophily appeared se
life (45). In light of the presence
topoisomerase domains in the d
evolution of hyperthermophily may
in agreement with the view of a h
Assuming that multidomain pro
of simple domains, then split gene
unit ancestral state of the proteins
may be split by DNA mutation, in
mosomal rearrangement events (4
spp., gene degradation has produ
Split genes
Table 1. Split noncontiguous genes in N. equitans
Gene
Large helicase-related protein
Topoisomerase I
DNA polymerase I*
Archaeosine tRNA–guanine
transglycosylase†
RNA polymerase subunit B‡
Glu–tRNAGln amidotransferase (gatE)
Reverse gyrase§
Hypothetical RNA-binding protein
Hypothetical protein
Alanyl–tRNA synthetase
CDS encoding CDS encoding
N-terminal part C-terminal part
NEQ003
NEQ045
NEQ068
NEQ124
NEQ409
NEQ324
NEQ528
NEQ305
NEQ173
NEQ245
NEQ434
NEQ438
NEQ495
NEQ547
NEQ156
NEQ396
NEQ318
NEQ506
NEQ096
NEQ211
*Also split in Methanothermobacter thermautotrophicus.
†Also split in Methanopyrus kandleri, the Methanosarcinales, A. fulgidus, the
extreme halophiles and crenarchaea.
‡Also split in methanogens, A. fulgidus, and extreme halophiles.
§Also split in Methanopyrus kandleri (different site).
equitans, the split sites for most of these genes lie between
functional domains of the encoded proteins; thus, it seems likely
that the two separated genes are expressed to form subunits of
a functional enzyme. The genes for two subunits of alanyl–tRNA
encodes the F and G motifs. Toge
although the two genes are separ
mosome. We predict that the two p
are expressed separately and then
reassembled intein has been excis
nism, as observed in the DnaE p
(42). Topoisomerase
Already
known to be I and revers
contain
inteins
in some archaea; h
split
in other
organisms
were detected in these split genes
The N. equitans reverse gyrase i
Splits occur in
encoding a helicase (NEQ434) and
domain boundaries
domain.
Reverse gyrase appears t
helicase and a topoisomerase do
supercoil formation in DNA (43
enzyme is present only in hyperth
that hyperthermophily appeared se
life (45). In light of the presence
topoisomerase domains in the d
evolution of hyperthermophily may
in agreement with the view of a h
Assuming that multidomain pro
of simple domains, then split gene
unit ancestral state of the proteins
may be split by DNA mutation, in
mosomal rearrangement events (4
spp., gene degradation has produ
Split genes
Table 1. Split noncontiguous genes in N. equitans
Gene
Large helicase-related protein
Topoisomerase I
DNA polymerase I*
Archaeosine tRNA–guanine
transglycosylase†
RNA polymerase subunit B‡
Glu–tRNAGln amidotransferase (gatE)
Reverse gyrase§
Hypothetical RNA-binding protein
Hypothetical protein
Alanyl–tRNA synthetase
CDS encoding CDS encoding
N-terminal part C-terminal part
NEQ003
NEQ045
NEQ068
NEQ124
NEQ409
NEQ324
NEQ528
NEQ305
NEQ173
NEQ245
NEQ434
NEQ438
NEQ495
NEQ547
NEQ156
NEQ396
NEQ318
NEQ506
NEQ096
NEQ211
*Also split in Methanothermobacter thermautotrophicus.
†Also split in Methanopyrus kandleri, the Methanosarcinales, A. fulgidus, the
extreme halophiles and crenarchaea.
‡Also split in methanogens, A. fulgidus, and extreme halophiles.
§Also split in Methanopyrus kandleri (different site).
equitans, the split sites for most of these genes lie between
functional domains of the encoded proteins; thus, it seems likely
that the two separated genes are expressed to form subunits of
a functional enzyme. The genes for two subunits of alanyl–tRNA
encodes the F and G motifs. Toge
although the two genes are separ
mosome. We predict that the two p
are expressed separately and then
reassembled intein has been excis
nism, as observed in the DnaE p
Trans-splicing
intein? I and revers
(42).
Topoisomerase
contain inteins in some archaea; h
were detected in these split genes
The N. equitans reverse gyrase i
encoding a helicase (NEQ434) and
domain. Reverse gyrase appears t
helicase and a topoisomerase do
supercoil formation in DNA (43
enzyme is present only in hyperth
that hyperthermophily appeared se
life (45). In light of the presence
topoisomerase domains in the d
evolution of hyperthermophily may
in agreement with the view of a h
Assuming that multidomain pro
of simple domains, then split gene
unit ancestral state of the proteins
may be split by DNA mutation, in
mosomal rearrangement events (4
spp., gene degradation has produ
Intein splicing
Intein splicing in trans?
intein
AB FG
mRNA
pre-protein
AB
FG
mRNA 1
mRNA 2
pre-protein 2
pre-protein 1
pre-protein
intein
mature protein
intein
mature protein
provided the opportunity to test the idea that the individual
protein parts are catalytically inactive, but that they reconstitute
activity when combined (41). Only a combination of both parts
of the split protein yielded a fully active enzyme as checked by
the standard aminoacylation assay (Fig. 2); thus, in this case,
covalent linkage is not a prerequisite for enzyme activity.
Many archaeal DNA processing and replication genes contain
inteins, intervening protein sequences that self-splice from nascent polypeptides. A split gene with remnants of an intein
encodes the N. equitans DNA-directed polymerase I (Table 1).
The C-terminal part of NEQ068 contains the A and B motifs for
protein cleavage, whereas the N-terminal region of NEQ528
Split genes
Table 1. Split noncontiguous genes in N. equitans
Gene
Large helicase-related protein
Topoisomerase I
DNA polymerase I*
Archaeosine tRNA–guanine
transglycosylase†
RNA polymerase subunit B‡
Glu–tRNAGln amidotransferase (gatE)
Reverse gyrase§
Hypothetical RNA-binding protein
Hypothetical protein
Alanyl–tRNA synthetase
CDS encoding CDS encoding
N-terminal part C-terminal part
NEQ003
NEQ045
NEQ068
NEQ124
NEQ409
NEQ324
NEQ528
NEQ305
NEQ173
NEQ245
NEQ434
NEQ438
NEQ495
NEQ547
NEQ156
NEQ396
NEQ318
NEQ506
NEQ096
NEQ211
encodes the F and G motifs. Toge
although the two genes are separ
mosome. We predict that the two p
are expressed separately and then
reassembled intein has been excis
nism, as observed in the DnaE p
(42). Topoisomerase I and revers
contain inteins in some archaea; h
were detected in these split genes
The N. equitans reverse gyrase i
encoding a helicase (NEQ434) and
domain. Reverse gyrase appears t
helicase and a topoisomerase do
supercoil formation in DNA (43
Functional
in 2 pieces
enzyme is present
only in hyperth
that hyperthermophily appeared se
life (45). In light of the presence
topoisomerase domains in the d
evolution of hyperthermophily may
in agreement with the view of a h
Assuming that multidomain pro
of simple domains, then split gene
unit ancestral state of the proteins
may be split by DNA mutation, in
mosomal rearrangement events (4
spp., gene degradation has produ
Fig. 2. Alanylation of unfractionated M. jannaschii tRNA by alanyl–tRNA
synthetases. The purification and aminoacylation procedures were adapted
from Ahel et al. (22) and are detailed in Materials and Methods. The enzymes
used are M. jannaschii AlaRS (filled squares), N. equitans AlaRS1 N-terminal
part (open circles), N. equitans AlaRS2 C-terminal part (filled triangles), and N.
equitans AlaRS1 " AlaRS2 (filled circles).
Waters et al.
*Also split in Methanothermobacter thermautotrophicus.
†Also split in Methanopyrus kandleri, the Methanosarcinales, A. fulgidus, the
extreme halophiles and crenarchaea.
‡Also split in methanogens, A. fulgidus, and extreme halophiles.
§Also split in Methanopyrus kandleri (different site).
equitans, the split sites for most of these genes lie between
functional domains of the encoded proteins; thus, it seems likely
that the two separated genes are expressed to form subunits of
a functional enzyme. The genes for two subunits of alanyl–tRNA
contiguous
pseudogene
scattered ab
evidence of
preceded by
gions of the
their simila
structures a
whether th
conservatio
genes and a
mosome) su
stable comp
Fig. 3. Phylo
determined by
ribosomal pro
resamplings. T
amino acid po
A dangling thread:
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All 61 non-stop codons are used in the normal fashion, but 3 of
the required tRNAs (Glu, His, Trp) are apparently absent.
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The authors suggest several possible explanations:
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they may be unusual structurally & therefore not found in the usual way,
tRNA “fragments” in the genome that could be joined to create functional tRNAs,
they could be imported from the host
there could be unusual multifunctional tRNAs
anticodon modifications could re-code tRNAs
Scattered fragments are joined to produce
the functional tRNAs
Figure 1 Predicted split N. equitans tRNA genes. a, tRNA half genes. The archaeal RNA
polymerase III promoter consensus box A motif, the tRNA half genes (red) and intervening
reverse complementary sequences (blue) are indicated. The positions of the tRNA
representation of the genomic distribution of tRNA genes (indicated by the amino-acid
three-letter code) and tRNA half genes (5 indicates the 5 0 tRNA half gene, 3 indicates
the 3 0 tRNA half gene) identified by our search algorithm. c, The joined sequences of
Glu
Met
scripts of these tRNA half genes include the intervening complementary sequences at the position of separation. In addition, RT–
PCR of anchor-ligated tRNA (Fig. 2c) revealed that the primary
transcript of the 5 0 tRNAHis half terminates at the AT-rich region
following the complementary downstream sequence found in all
tRNA half genes.
tRNA gene fragments
circularization of the tRNA we were able to identify the 5 and 3
ends of the mature tRNA. Our sequencing results show sizematuration of the joined tRNAGlu, as a CCA sequence is indeed
added to the 3 0 end of both tRNAGlu isoacceptors after transcription
(Fig. 2b). A final requirement for tRNA functionality in vivo is the
ability to serve as a substrate for amino acid attachment by
letters to nature
The answer
- trans-splicing
contain leader sequences ups
absence, there would not be a n
organism.
An extensive search of t
genome sequences did not re
isms. Future sequences of ot
should reveal whether split t
genome2 or whether they are
size reduction. The sequencin
therefore eagerly awaited.
Methods
Computational method for tRNA id
Figure 4 Schematic representation of a 5 0 tRNA half gene (tRNAGlu) and the
corresponding 3 0 tRNA half gene found in N. equitans. The archaeal RNA polymerase III
promoter consensus sequence (TTTAAA), the tRNA half genes (red) and the intervening
reverse complementary sequences that are supposed to facilitate joining of the halves
(blue) are indicated.
tRNA genes were predicted by use of a
Virtual Footprint (http://www.prodoric
from both a conserved, continuous 3 0 r
tRNA genes (nt 1–16) in an alignment o
For this purpose the information conte
modifications. tRNA gene searches were
a genome scale with the highest sensitiv
scoring sequence of the training set). Th
stretch of 7 nt to a reverse complementa
was used to identify matching pairs of
previously annotated tRNAs were iden
fell into the threshold range of the ann
Cell culture and tRNA isolation
N. equitans cells were grown in a 300-l f
sp. and purified by gradient centrifuga
chemical digestion with 2% SDS, follow
described29. The tRNA was further pur
chromatography to eliminate residual
eluted with a linear 60-ml gradient of 0
L. Randau et al. / FEBS Letters 579 (2005) 2945–2947
2947
Fig. 3. The set of processed tRNAs of N. equitans. (A) Alignment of tRNAs with the genomic position indicated. The tRNA halves are joined at the
position indicated by a slash. Possible secondary structures of relaxed Bulge-Helix-Bulge motifs for (B) cis-spliced tRNAs and (C) joined (transspliced) tRNA half genes are given. The individual anticodons are boxed and the splice sites are indicated by arrows. The tRNA sequence is written in
uppercase letters and the excised intervening sequences are written in lowercase letters.
In organisms where a chromosome rearrangement
divided the intron, the resistance to integration would
be stabilized. Upon repeated exposure to an integrative element, trans-spliced tRNA genes could become fixed in the
[3] Huber, H., Hohn, M.J., Rachel, R., Fuchs, T., Wimmer, V.C. and
Stetter, K.O. (2002) A new phylum of Archaea represented by a
nanosized hyperthermophilic symbiont. Nature 417, 63–67.
[4] Freist, W., Gauss, D.H., Ibba, M. and Söll, D. (1997) Glutaminyl-
Another (unmentioned) dangling thread:
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No RNase P RNA gene, nor any of the 4 standard archaeal
RNase P protein genes.
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RNase P has been identified in everything except
Nanoarchaeum, Pyrobaculum, and Aquifex.
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RNase P is essential for tRNA biosynthesis
Possible explanations:
•
•
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Unusual “standard” RNase P subunits that evade recognition
A completely novel RNase P enzyme
tRNA precursors without 5´ leaders
mentary sequences at the position of separation. In addition, RT–
PCR of anchor-ligated tRNA (Fig. 2c) revealed that the primary
transcript of the 5 0 tRNAHis half terminates at the AT-rich region
following the complementary downstream sequence found in all
tRNA half genes.
No answer yet, but....
•
ends of the mature tRNA. Our sequencing results show sizematuration of the joined tRNAGlu, as a CCA sequence is indeed
added to the 3 0 end of both tRNAGlu isoacceptors after transcription
(Fig. 2b). A final requirement for tRNA functionality in vivo is the
ability to serve as a substrate for amino acid attachment by
It looks like pre-tRNAs may be transcribed directly at or near
their functional 5´ ends, & so RNase P may be dispensable.