88 231 Bacterial Transposons

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23.1 Bacterial Transposons
23.1 Bacterial Transposons
In transposition, a transposable element, or transposon,
moves from one DNA address to another. Barbara
McClintock discovered transposons in the 1940s in her
studies on the genetics of maize. Since then, transposons
have been found in all kinds of organisms, from bacteria
to humans. We will begin with a discussion of the bacterial
transposons.
Discovery of Bacterial Transposons
James Shapiro and others laid the groundwork for the discovery of bacterial transposons with their discovery in the
late 1960s of phage mutations that did not behave normally. For example, they did not revert readily the way
point mutations do, and the mutant genes contained long
stretches of extra DNA. Shapiro demonstrated this by taking advantage of the fact that a l phage will sometimes
pick up a piece of host DNA during lytic infection of E. coli
cells, incorporating the “passenger” DNA into its own genome. He allowed l phages to pick up either a wild-type
E. coli galactose utilization gene (gal1) or its mutant counterpart (gal2), then measured the sizes of the recombinant
DNAs, which contained l DNA plus host DNA. He measured the DNA sizes by measuring the densities of the two
types of phage using cesium chloride gradient centrifugation (Chapter 20). Because the phage coat is made of protein and always has the same volume, and because DNA is
much denser than protein, the more DNA the phage contains the denser it will be. It turned out that the phages
harboring the gal2 gene were denser than the phages with
the wild-type gene and therefore held more DNA. The simplest explanation is that foreign DNA had inserted into the
gal gene and thereby inactivated it. Indeed, later experiments revealed 800–1400-bp inserts in the mutant gal gene,
which were not found in the wild-type gene. In the rare
cases when such mutants did revert, they lost the extra
DNA. These extra DNAs that could inactivate a gene by
inserting into it were the first transposons discovered in
bacteria. They are called insertion sequences (ISs).
Insertion Sequences:
The Simplest Bacterial Transposons
Bacterial insertion sequences contain only the elements
necessary for transposition. The first of these elements is a
set of special sequences at a transposon’s ends, one of
which is the inverted repeat of the other. The second element is the set of genes that code for the enzymes that
catalyze transposition.
Because the ends of an insertion sequence are inverted
repeats, if one end of an insertion sequence is 59-ACCGTAG, the other end of that strand will be the reverse complement: CTACGGT-39. The inverted repeats given here
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are hypothetical and are presented to illustrate the point.
Typical insertion sequences have somewhat longer inverted
repeats, from 15 to 25 bp long. IS1, for example, has inverted repeats 23 bp long. Larger transposons can have
inverted repeats hundreds of base pairs long.
Stanley Cohen provided one graphic demonstration of
inverted repeats at the ends of a transposon with the experiment illustrated in Figure 23.1. He started with a plasmid containing a transposon with the structure shown on
the left in Figure 23.1a. The original plasmid was linked to
the ends of the transposon, which were inverted repeats.
Cohen reasoned that if the transposon really had inverted
repeats at its ends, he could separate the two strands of the
recombinant plasmid, and get the inverted repeats on one
strand to base-pair with each other, forming a stem-loop
structure as shown on the right in Figure 23.1a. The stems
would be double-stranded DNA composed of the two
inverted repeats: the loops would be the rest of the DNA
in single-stranded form. The electron micrograph in Figure 23.1b shows the expected stem-loop structure.
The main body of an insertion sequence codes for at
least two proteins that catalyze transposition. These proteins
are collectively known as transposase; we will discuss their
mechanism of action later in this chapter. We know that
these proteins are necessary for transposition because
mutations in the body of an insertion sequence can render
that transposon immobile.
One other feature of an insertion sequence, shared
with more complex transposons, is found just outside the
transposon itself. This is a pair of short direct repeats in
the DNA immediately surrounding the transposon. These
repeats did not exist before the transposon inserted; they
result from the insertion process itself and tell us that the
transposase cuts the target DNA in a staggered fashion
rather than with two cuts right across from each other.
Figure 23.2 shows how staggered cuts in the two strands
of the target DNA at the site of insertion lead automatically to direct repeats. The length of these direct repeats
depends on the distance between the two cuts in the target
DNA strands. This distance depends in turn on the nature
of the insertion sequence. The transposase of IS1 makes
cuts 9 bp apart and therefore generates direct repeats that
are 9 bp long.
SUMMARY Insertion sequences are the simplest of
the bacterial transposons. They contain only the elements necessary for their own transposition; short
inverted repeats at their ends and at least two genes
coding for an enzyme called transposase that carries
out transposition. Transposition involves duplication of a short sequence in the target DNA; one
copy of this short sequence flanks the insertion
sequence on each side after transposition.
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GTC
TG
GG
Internal genes
Inverted terminal repeats
GTC
AC
C AG
CAG TGGG
CC GTCTG
AC
CC
GG
Transposable element
Strand separation
Intra-strand annealing
CAG
AC
CC
Plasmid DNA
CAGACCC
TG
GTC
GG
GGGTCTG
Inverted terminal repeats
GTCTGGG
AC
CAG
CC
(a)
CCCAGAC
Chapter 23 / Transposition
(b)
Figure 23.1 Transposons contain inverted terminal repeats.
(a) Schematic diagram of experiment. The two strands of a transposonbearing plasmid were separated and allowed to anneal with themselves
separately. The inverted terminal repeats will form a base-paired stem
between two single-stranded loops corresponding to the internal
genes of the transposon (small loop, green) and host plasmid (large
loop, purple and pink). (b) Experimental results. The DNA was
shadowed with heavy metal and subjected to electron microscopy.
The loop-stem-loop structure is obvious. The stem is hundreds of base
pairs long, demonstrating that the inverted terminal repeats in this
transposon are much longer than the 7 bp shown for convenience in
part (a). (Source: (b) Courtesy Stanley N. Cohen, Stanford University.)
More Complex Transposons
and growing these host bacteria in medium containing
both antibiotics. If the bacteria survive, they must have
taken up both antibiotic resistance genes; therefore, Tn3
must have transposed to the target plasmid.
Insertion sequences and other transposons are sometimes
called “selfish DNA,” implying that they replicate at the
expense of their hosts and apparently provide nothing useful in return. However, some transposons do carry genes
that are valuable to their hosts, the most familiar being
genes for antibiotic resistance. Not only is this a clear benefit to the bacterial host, it is also valuable to molecular
biologists, because it makes the transposon much easier to
track.
For example, consider the situation in Figure 23.3, in
which we start with a donor plasmid containing a gene for
kanamycin resistance (Kanr) and harboring a transposon
(Tn3) with a gene for ampicillin resistance (Ampr); in addition, we have a target plasmid with a gene for tetracycline
resistance (Tetr). After transposition, Tn3 has replicated
and a copy has moved to the target plasmid. Now the
target plasmid confers both tetracycline and ampicillin
resistance, properties that we can easily monitor by transforming antibiotic-sensitive bacteria with the target plasmid
Mechanisms of Transposition
Because of their ability to move from one place to another,
transposons are sometimes called “jumping genes.” However, the term is a little misleading because it implies that
the DNA always leaves one place and jumps to the other.
This mode of transposition does occur and is called nonreplicative transposition (or “cut and paste”) because both
strands of the original DNA move together from one place
to the other without replicating. However, transposition
frequently involves DNA replication, so one copy of the
transposon remains at its original site as another copy inserts at the new site. This is called replicative transposition
(or “copy and paste”) because a transposon moving by this
route also replicates itself. Let us discuss how both kinds of
transposition take place.
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CATAGCCTGAT
GTATCGGACTA
Target plasmid
Cut at arrows
CATAGCCTGA
G
(b)
T
TATCGGACTA
Insert transposon
(c)
CATAGCCTGA
G
T
TATCGGACTA
Fill in gaps
(d)
C ATAGCCTGA
G TATCGGACT
ATAGCCTGA T
TATCGGACT A
Figure 23.2 Generation of direct repeats in host DNA flanking a transposon. (a) The arrows indicate where the two strands of host DNA will be
cut in a staggered fashion, 9 bp apart. (b) After cutting. (c) The transposon (yellow) has been ligated to one strand of host DNA at each end, leaving
two 9-bp base gaps. (d) After the gaps are filled in, 9-bp repeats of host DNA (pink boxes) are apparent at each end of the transposon.
Ampr
tnpA
IR
Tn3
res
bla
IR
Figure 23.4 Structure of Tn3. The tnpA and tnpR genes are necessary
for transposition; res is the site of the recombination that occurs during
the resolution step in transposition; the bla gene encodes b-lactamase,
which protects bacteria against the antibiotic ampicillin. This gene is
also called Ampr. Inverted repeats (IR) are found on each end. The
arrows indicate the direction of transcription of each gene.
r
Tet
r
Kan
Transposition
Ampr
tnpR
Ampr
r
r
Kan
Tet
Figure 23.3 Tracking transposition with antibiotic resistance
genes. We begin with two plasmids: The larger (blue) encodes
kanamycin resistance (Kanr ) and bears the transposon Tn3 (yellow),
which codes for ampicillin resistance (Ampr); the smaller (green)
encodes tetracycline resistance (Tetr ). After transposition, the smaller
plasmid bears both the Tetr and Ampr genes.
Replicative Transposition of Tn3 Tn3, whose structure is shown in Figure 23.4, illustrates one well-studied
mechanism of transposition. In addition to the bla gene,
which encodes ampicillin-inactivating b-lactamase, Tn3
contains two genes that are instrumental in transposition.
Tn3 transposes by a two-step process, each step of which
requires one of the Tn3 gene products. Figure 23.5 shows
a simplified version of the sequence of events. We begin
with two plasmids; the donor, which harbors Tn3, and the
target. In the first step, the two plasmids fuse, with Tn3
replication, to form a cointegrate in which they are
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Chapter 23 / Transposition
Transposon
Target
Target
Transposon
tnpA fusion
(Step 1) Nicking DNA
e f
a b
Cointegrate
cd
tnpR resolution
gh
(Step 2) Joining free ends
Figure 23.5 Simplified scheme of the two-step Tn3 transposition.
In the first step, catalyzed by the tnpA gene product, the plasmid
(black) bearing the transposon (blue) fuses with the target plasmid
(green, target in red) to form a cointegrate. During cointegrate
formation, the transposon replicates. In the second step, catalyzed by
the tnpR gene product, the cointegrate resolves into the target
plasmid, with the transposon inserted, plus the original transposonbearing plasmid.
coupled through a pair of Tn3 copies. This step requires
recombination between the two plasmids, which is catalyzed by the product of the Tn3 transposase gene tnpA.
Figure 23.6 shows a detailed picture of how all four DNA
strands involved in transposition might interact to form
the cointegrate. Figures 23.5 and 23.6 illustrate transposition between two plasmids, but the donor and target
DNAs can be other kinds of DNA, including phage DNAs
or the bacterial chromosome itself.
The second step in Tn3 transposition is a resolution of
the cointegrate, in which the cointegrate breaks down into
two independent plasmids, each bearing one copy of Tn3.
This step, catalyzed by the product of the resolvase gene
tnpR, is a recombination between homologous sites on
Tn3 itself, called res sites. Several lines of evidence show
that Tn3 transposition is a two-step process. First, mutants
in the tnpR gene cannot resolve cointegrates, so they cause
formation of cointegrates as the final product of transposition. This demonstrates that the cointegrate is normally an
intermediate in the reaction. Second, even if the tnpR gene
is defective, cointegrates can be resolved if a functional
tnpR gene is provided by another DNA molecule—the host
chromosome or another plasmid, for example.
Nonreplicative Transposition Figures 23.5 and 23.6 illustrate the replicative transposition mechanism, but transposition does not always work this way. Some transposons
(e.g., Tn10) move without replicating, leaving the donor
DNA and appearing in the target DNA. How does this occur? It may be that nonreplicative transposition starts out
in the same way as replicative transposition, by nicking and
joining strands of the donor and target DNAs, but then
something different happens (Figure 23.7). Instead of replication occurring through the transposon, new nicks appear
(Step 3) Replication of transposon
af
h
c
af h
c
e
b
e
gd
b
d
g
(Step 4)
Completion
of replication
b
e gd
af
c
h
=
(Step 5)
Recombination
between res sites
(Step 6)
+
Figure 23.6 Detailed scheme of Tn3 transposition. Step 1: The two
plasmids are nicked to form the free ends labeled a–h. Step 2: Ends a
and f are joined, as are g and d. This leaves b, c, e, and h free. Step 3:
Two of these remaining free ends (b and c) serve as primers for DNA
replication, which is shown in a blowup of the replicating region. Step 4:
Replication continues until end b reaches e and end c reaches h. These
ends are ligated to complete the cointegrate. Notice that the whole
transposon (blue) has been replicated. The paired res sites (purple) are
shown for the first time here, even though one res site existed in the
previous steps. The cointegrate is drawn with a loop in it, so its derivation
from the previous drawing is clearer; however, if the loop were opened
up, the cointegrate would look just like the one in Figure 23.5 (shown
here at right). Steps 5 and 6 (resolution): A crossover occurs between the
two res sites in the two copies of the transposon, leaving two independent
plasmids, each bearing a copy of the transposon.