Complications and implications
of linear bacterial chromosomes
C. W. Chen
Trends in Genetics 12: 192-196
(1996)
The emergence of linear bacterial chromosomes
has overthrown the dogma of universal circularity of the bacterial
chromosomes, and posed mechanistic and evolutionary implications
not previously anticipated. These are the subject of this article.
The first linear bacterial chromosome,
that of the spirochete Borrelia
burgdorferi, was readily detected
by standard pulsed-field gel electrophoresis technique due to
its relatively small size (960 kb)1. Then, there was evidence
from physical mapping that one linear and one circular chromosome
exist in Agrobacterium tumefaciens2. For the chromosomes of gram-positive bacteria Streptomyces, which are among the largest in bacteria (about 8 Mb), discovering
and proving their linearity was significantly more difficult3. It was achieved first in Streptomyces lividans 66, and later all other species investigated3-6. Rhodococcus fascians,
another member of the actinomycetes, also appeared to have a linear
chromosome7. Application of the powerful new tools are likely to uncover
linear chromosomes in more bacteria. The linear chromosomes of
Streptomyces (particularly that of S. lividans), being best characterized,
will be used to exemplify the discussion hereafter.
Linear bacterial chromosomes are often
accompanied by linear plasmids. All the linear chromosomes and
plasmids of Streptomyces characterized
appear to contain terminal inverted repeat (TIR) and covalently-bound
terminal proteins (TP). Although these features are shared by
a group of linear replicons including some viral and plasmid DNAs,
of which the most studied cases are adenoviruses and bacteriophage
[phi]29 of Bacillus subtilis (reviewed in Ref. 8), there is at least one basic difference:
while replication of adenoviruses and [phi]29 DNA is initiated
at the end (using the TP as the primer) and proceeds through the
whole genome to the other end, replication of linear plasmids9
and chromosomes10 of Streptomyces starts at an internal origin.
The sizes of the TIR range widely on the
linear plasmids (0.04 to 80 kb)11,
12, and linear chromosomes
(24 to 210 kb)3, 5, 6 in Streptomyces. At least one linear
plasmid (SLP2 in S. lividans)
shares a long (16 kb) terminal sequence with its chromosomal counterpart3, 11. The terminal sequence
of the S. lividans
chromosome is rich in palindromes (four elements each of 13-23
bp including a 3 bp gap, in the first 90 bp), a feature shared
by the termini of the linear plasmids4 and chromosomes (C.-H. Huang and C. W. Chen, unpublished). A
comparison of these terminal sequences reveals a strong tendency
to preserve palindromicity: a base substitution in one repeat
is usually accompanied by a complementary substitution in its
partner sequence. Protein-binding palindromes frequently tolerate
imperfection in the repeats, so the persistent palindromicity
may reflect a different function (see later).
Linear plasmids of Borrelia, like those of Streptomyces, also range widely in size -- from 5 kb to > 200 kb. The termini
of a 16 kb linear plasmid of B. burgdorferi contain a perfectly
palindromic A+T-rich hairpin loop and a TIR13, a feature shared by other Borrelia linear plasmids. The
termini of the Borrelia chromosomes
have not been isolated, and their structures and sequences are
unknown. Genetic organization resembling that of a typical bacterial
oriC was also located in the
middle of the B. burgdorferi
chromosome.
Complications in replication telomere
patching
Perhaps the most prominent issue is how
a linear bacterial chromosome is replicated. The replication origin
(oriC) of Streptomyces14, 15
and the putative oriC
of Borrelia 16, 17 resemble those in other
eubacteria in genetic arrangement, and there is no reason to doubt
that they share the basic mechanism and regulation. The main issue
is at the termini, which normal replicating machinery cannot fully
replicate18.
Shiffman and Cohen19 showed that the linear plasmid
pSCL of S. clavuligerus
could replicate as a circular DNA molecule when the telomeres
were removed, indicating the existence of an internal origin of
replication within this linear replicon. Chang and Cohen9 further showed that replication
of the linear plasmid pSLA2 of Streptomyces rochei proceeds
from an internal origin toward the termini, leaving a 280-nt single-strand
gap at the 3' end, which was supposedly patched up by TP-primed
replication. Musialowski et al.10 showed evidence for replication
of S. coelicolor
initiated at oriC
ran bi-directionally toward the telomeres. This would also leave
a single-stranded gap at each end (Fig. 1a). How are the gaps
filled?
FIGURE 1. Models for patching the telomeres of Streptomyces plasmids and chromosomes. (a) The putative telomere structure
after replication. The upper daughter DNA is fully duplicated,
whereas the lower one remains single-stranded at the 3' end. The
four terminal palindromes (I - IV) in S. lividans are indicated. (b)
The unlikely mechanism of patching replication by TP and DNA polymerase
initiated at the single-strand 3' end. (c) Alternative model 1:
TP and DNA polymerase recognize and initiate replication at the
duplex formed by foldback of the protruding 3' end. (d) Alternative
model 2: terminal initiation occurs at the fully duplex daughter
DNA, displacing the TP-capped 5' strand, which is transferred
by homologous recombination to the terminal 3' gap on the sibling
chromosome. (e) Alternative model 3: the terminal palindrome of
the protruding 3' end folds back and provides a primer for the
patching synthesis, followed by nicking by TP and fill-in synthesis
that restores the telomere. See text for details. A straight-forward
solution is for DNA polymerase to start replication at the telomere
using TP as the primer (Fig. 1b). There is, however, an important
difference between this scheme and the replication of adenoviruses
and [phi]29: The signal for the initiation of terminal replication
in adenoviruses and [phi]29 is double-strand DNA, which provides
sequence-specific structural information for recognition by TP
and DNA polymerase8. Such a stable conformation is unlikely to be achieved by the
single-strand terminus on the Streptomyces chromosomes in the form of a random coil. Other features of the
terminal DNA may be invoked.
There are at least three alternatives.
In the first model (Fig. 1c; initially suggested by S. N. Cohen),
the protruding 3' end folds back and pairs with an internal sequence.
The abundance and persistence of palindromic sequences in the
terminal regions support this view. For example, the terminal
13 bases (palindrome I) of the S.
lividans chromosome may fold
back and pair with a complementary sequence in palindrome IV (59
bp away)4. Formation of the I-IV duplex may be aided by fold-back of the
intervening palindromes II and III. A patching replication is
initiated at the I-IV duplex by the TP and polymerase and proceeds
inward along the single-strand 3' end.
In the second model (Fig. 1d), the TP primes
replication at the telomere of the fully duplicated daughter DNA,
displacing the parental 5' strand (with the attached TP), which
then pairs with the protruding 3' end. Strand exchange and resolution
of the half-Holiday junction is then carried out by homologous
recombination. This model, therefore, suggests an essential role
of recA
(for strand invasion) and ruv
genes (for resolution) in cell
viability. Indeed, there has been unexpected difficulty in generating
null mutations of recA in Streptomyces using conventional gene targeting procedures (A.-J Cheng, S.-T.
Hu, C. W. Chen, unpublished results; W. Wohlleben, personal communication).
Moreover, strand exchange in this model may be across two arms
of the replicon, which would allow homogenotization of the terminal
sequences or repair of a damaged telomere through gene conversion.
In the third model (Fig. 1e), palindrome
I folds back on itself to form a hairpin such that its 3' end
serves as the primer for gap filling replication. The TP nicks
the resulting hairpin at a site opposite the original 3' end,
and becomes covalently bound to the 5' end of the nicked strand.
Further extension synthesis from the new 3' end at the nick unfolds
the hairpin and restores the telomere structure. This mode of
terminal patching is analogous to the 'rolling-hairpin' model
for replication of parvovirus DNA18, in which a viral-encoded
protein carries out site-specific incision while remaining covalently
linked to the 5' end. This type of endonucleolytic protein also
includes the gA protein of [phi]X174 phage, the g2 protein of
Ff phage and the TraI protein of F plasmid. In this model the
chromosomal TP has a different role than the replication-priming
TPs of adenoviruses and [phi]29. Moreover, this model predicts
that each round of replication will flip the terminal palindrome
sequence once. This leads to a testable prediction that, among
the population of S. lividans
chromosomes, the imperfect Palindrome
I exists in both orientations. This kind of 'hairpin transfer'
has been demonstrated in parvovirus replication. The linear chromosomes
of Borrelia, if they contain hairpin
termini like their linear plasmid counterparts, may use the same
mechanism proposed for other hairpin-capped DNA such as poxvirus
and ridovirus20, which also shares the 'hairpin
transfer' mechanism although the endonuclease does not remain
covalently attached.
Complications in replicative transposition
unresolvable cointegrates
Replicative transposition between linear
bacterial replicons also has intriguing complications. In the
traditional model built on circular replicons, replicative transposition
generates a circular cointegrate molecule containing donor and
target molecules separated by two directly repeated copies of
the transposable element (Fig. 2a). For one class (such as Mu
phage of E. coli), there is no resolution system, and the cointegrate is the final
product. For another class of transosable elements typified by
the Tn3 family, the cointegrate is resolved by an element-specific resolvase
that aligns the resolution (res) sequences on the elements and carries out site-specific recombination21. There is a barrier to intermolecular
resolution (leading to fusion of the circular replicons); no such
reaction has been reported.
Application of these models of replicative
transposition to linear replicons present a problem. While the
recipient DNA would receive a new copy of the transposable element,
two separate molecules (instead of a fused cointegrate) would
be produced, which are unlikely to be 'resolved' even with a resolution
system (Fig. 2b). Thus, the two recombinat replicons would conceivably
remain the final product. In such transposition between a linear
chromosome and a linear plasmid, a segment of the chromosome would
be transferred to the plasmid. This may be a serious hazard if
the lost segment contains essential genes. If the transposition
is between two chromosomal DNA molecules, the process is similar
to an unequal crossover, producing a deletion in one chromosome
and an insertion in the other. The scarcity of important genes
in the terminal regions of the Streptomyces chromosomes3 implies that if replicative transposition is not to produce deletions
that have serious effects, it could be targeted only to the terminal
regions of the chromosome. In this regard, the existence of the
partial SLP2 DNA segment at the end of the S.
lividans chromosome bordered by a transposon11 (that is not present elsewhere
in the chromosome) resembles such a transposition. The recent
observation that a linear plasmid appears to integrate and replace
the end of the Streptomyces rimosus
chromosome22 offers
an new opportunity to test the model.
FIGURE 2. Replicative transposition and replicon topology. (a) Circular replicons. Replicative transposition gives rise to a cointegrate,
in which the two replicons are fused by the duplicated copies
of transposable element. The element-specific resolvase aligns
the res sites and resolves the two replicons by site-specific recombination.
The continuity of the cointegrate molecule is important for the
resolution reaction. (b) Linear replicons. Replicative transposition creates two recombinant
replicons, each with a copy of the transposable elements at the
junction of the exchange. This discontinuous 'cointegrate' may
be the final product, because resolution is unlikely (shown in
parentheses) in the absence of continuity between the two molecules,
according to the current model.
Complications in conjugal transfer the
terminal enigma
Conjugal transfer of plasmids or chromosomes
has also been modeled on circular replicons18. In the classical model
of the F plasmid of E. coli, transfer is initiated
by nicking at a specific site (oriT) on the plasmid, followed
by rolling-circle replication (Fig. 3a). The displaced 5' strand,
covalently linked to a plasmid-specific transfer protein (TraI),
is transferred to the recipient cell. There is no basic biochemical
difference between transfer of the plasmid (F) and the plasmid-chromosome
cointegrate (Hfr), except for their sizes.
A biochemically analogous model for the
transfer of linear Streptomyces
plasmids and chromosomes is for
TP-primed replication to initiate at the telomere (oriT) and displace the parental
5' TP-capped strand into the recipient (Fig. 3b). This proposed
conjugal replication, unlike the proposed terminal patching (Fig.
1), is initiated at the fully duplexed terminus. The conjugal
replication proceeds along the entire chromosome, preempting the
normal bi-directional replication as shown in E. coli. Moreover, a linear plasmid
may provide trans-acting functions for the
mobilization of a chromosome without physical integration. This
agrees with the recent observation of Pettis and Cohen23 that chromosomal gene transfer in S. lividans required trans-acting but not cis-acting plasmid functions.
FIGURE 3. Conjugal transfer of circular and linear replicons. (a) Circular
plasmid (F) and circular chromosome (Hfr) in E.
coli: in both cases, F-specific proteins carry out a nicking-priming
event at oriT on F DNA, where the 5' end
is covalently attached to one of the transfer proteins (TraI),
and a rolling circle replication is initiated at the 3' end. The
protein-capped 5' strand is continuously displaced and transferred
to the recipient cell. In Hfr cells, the chromosomal DNA is led
by the F sequence into the recipient cell as an exogenote. (b)
Linear plasmids and linear chromosomes of Streptomyces. The TP-capped ends
may act as the origin of transfer (oriT), where the putative TP-primed
replication is initiated, and the 5' TP-bound strand is displaced
and transferred, as for F/Hfr transfer.
Conclusions
This article raises some of the issues
inherent in the linearity of bacterial replicons, and offers models
that suggest future investigations. It echoes the sentiment that
what is true for E. coli may not necessarily be true for other bacteria. At present we
know neither how many forms of linear chromosomes there are, nor
how the eubacterial kingdom is divided with respect to chromosomal
topology. Each type of chromosome may occupy a distinct taxonomic
domain, which would imply that chromosome topology plays an important
role in the evolution of bacteria. On the other hand, it is also
possible that topological conversion is frequent, and that both
linear and circular chromosomes exist in closely related bacteria.
The long observed structural instability (extensive deletions
and amplifications) of Streptomyces
chromosomes has been recently
attributed to rearrangements at the terminal regions, some of
which even lead to circularization of the chromosome3. The significance of the
structural instability of Streptomyces chromosomes has recently been discussed elsewhere24. It remains to be seen whether the topological conversion observed
in Streptomyces3, 19
and mitochondria25 has an evolutionary role.
Acknowledgments
This work is supported by research grants
(NSC84-0418-B010-033-BC, NSC85-2331-B010-005-BC) from National
Science Council, Taiwan. I thank Drs. David A. Hopwood and Stanley
N. Cohen for critical reading and helpful comments on the manuscript.
This paper is dedicated to the memory of my father, C.-C. Chen.
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