bacterial transposon tn5: evolutionary inference8

8/20/2019 Bacterial Transposon Tn5: Evolutionary Inference8

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Bacterial Transposon Tn5: Evolutionary Inference8

Douglas E. Berg,* Claire M. Berg,? and Chihiro Sasakawa*

*Departments

of

Microbiology

and Immunology,

Washington University School of Medicine

and of Genetics,

tBiologica1 Sciences Group, University of Connecticut

Transposable elements induce spontaneous mutations, promote genome rear-

rangements, regulate gene expression, and participate in the horizontal spread of

genes encoding traits such as antibiotic resistance among bacterial genera too

distantly related to undergo homologous recombination. Here we review the bacterial

transposon Tn5 and focus on those aspects of its functional organization and

transposition which provide insights into how it and other elements may have

arisen, proliferated, and evolved.

Introduction

Transposable elements are discrete DNA segments that exhibit the specialized

ability to move from site to site in a genome independent of extensive DNA sequence

homology. Their existence was first documented by McClintock in her prescient

genetic analyses of unstable alleles in maize. More recently the existence and importance

of transposable elements has been established in many groups of organisms. As

McClintock first showed, transposable elements often cause spontaneous mutations,

regulate the expression of genes near their insertion sites, and induce cycles of chro-

mosome breakage and rearrangement. Transposition-like phenomena are also im-

plicated in the DNA rearrangements that occur during the normal development of

the immune system and in the unscheduled chromosomal translocations associated

with many types of cancer (for reviews see Shapiro [ 19831).

Transposable elements play a special role in bacterial evolution because of their

ability to move between the chromosome and the various plasmid and temperate

phage DNAs resident in a bacterial cell and, when piggybacked on these molecules,

to move between unrelated bacteria in a population. Virtually any gene can become

associated with a transposable element, and complex elements called transposons

containing genes whose functions are unrelated to movement are now commonplace.

Those that encode resistance to clinically useful antibiotics have been studied most

intensively because their resistance determinants have provided selectable genetic

markers that are valuable in basic studies of transposition mechanisms and in the

use of transposons as mutagens in many bacterial species, and because the epidemic

spread of antibiotic resistance among bacterial populations has severely compromised

strategies for the treatment and prevention of infectious disease. Consideration of the

clonal nature of most bacterial growth, the participation of transposable elements in

1. Key words: insertion sequences, antibiotic resistance, interspecific gene exchange, mechanism of

transposition, DNA sequence recognition.

Current address: Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108, Japan.

Address for correspondence and reprints: Douglas E. Berg, Department of Microbiology and Im-

munology, Box 8093, Washington University School of Medicine, 4566 Scott Avenue, St. Louis, Missouri

63110.

Mol. Biol. Evol 1(5):41 l-422. 1984.

0 1984 by The University of Chicago. All rights reserved.

0737~4038/84/0105-0003 02.00

411


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4

12 Berg, Berg, and Sasakawa

a network of exchange that transcends classical barriers between species and genera,

and the rapidity of the spread of resistance following the onset of routine usage of

antibiotics in medicine and agriculture suggest that it is especially during periods of

drastic environmental change that transposable elements make their greatest contri-

butions to the adaptability and evolution of bacterial populations.

How transposable elements have evolved is of great interest, and insights into

their evolution are emerging from analyses in bacteria of their functional organization,

and the mechanism and regulation of their movement. Among the best known of

the procaryotic elements is the kanamycin resistance transposon Tn5, and it is on

this element that the following discussion focuses.

Transposon Tn5

Functional Organization

Tn5 is a 5,700-base pair (bp) composite element in which a pair of simpler

mobile elements, the 1,534-bp insertion sequences ISSOR and ISSOL, are present in

inverted orientation and bracket a central region that contains genes encoding resistance

to kanamycin

kan)

and to streptomycin (str; fig. 1; for review, Berg and Berg [ 19831).

Tn5 was discovered as a component of a bacterial R factor plasmid. It exhibits no

significant similarity to the half-dozen transposable elements indigenous to the genome

of Escheri chi a coli K-12 or to sequences in the vast majority (>95%) of several

hundred enteric isolates that have been screened. Tn5 transposes with high frequency

and inserts into many sites, including into a small number of hot spots. When inserted

into an operon, Tn5 blocks the normal transcription of distal genes. At certain sites,

however, it causes low-level constitutive expression of distal genes. Like several of

the other bacterial elements, Tn5 generates a direct 9-bp duplication of the target

sequence at its site of insertion, probably reflecting staggered cuts made in the target

DNA during transposition and limited repair synthesis to fill in the resulting gap.

SOL p

kan’ (str’ )*

SSOR

‘ I

runcated

ochre

proteins

w transposase -

+ inhibitor+

HgaI

Pvu IL BglII BCII

1521

I

C TGACTC TTATACACAAGT;....

GACTGAGAATATGTGTTCAT....

--

0 End

. . ..iAGATCTGATCAAGAGAC

. . ..TTCTAGACTAGTTCTCTGTC

transpasase gene

4

GA

I End

stop

FIG. 1

-Top,

Functional organization of transposon Tn5 Rothstein and Reznikoff 1981; Berg et al.

1982). The gene encoding resistance to streptomycin is indicated in parentheses because its transcript is

not translated in Escherichia

coli

Putnoky et al. 1983; Selvaraj and Iyer 1984).

Bottom,

The structure of

ISSOR, showing the DNA sequence of its essential transposase recognition sites. ISSOL s identical except

for a single base pair change 112 bp from the inside end Auerswald et al. 1980; Isberg et al. 1982; Johnson

et al. 1982; Sasakawa et al. 1983). 0 = outside; I = inside.


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Transposon Tn5 4 13

Of the two IS elements in Tn5, ISSOR encodes a cis-acting protein, transposasc,

that is necessary for IS50 and Tn5 movement (Isberg and Syvanen 198 1; Rothstein

and Reznikoff 198 1). Transposase is thought to act by binding distinctive 19-bp

nucleotide sequences near the ends of its cognate (IS50) elements (Johnson and

Reznikoff 1983; Sasakawa et al. 1983). ISSOL differs from ISSOR in that it contains

the promoter used for expression of the

kan

gene in TnS’s central region, and also

an ochre allele of the transposase

tnp)

gene. Both the

kan

promoter and the mutant

tnp allele arise from a substitution of one nucleotide pair 112 bp from ISSo’s inside

end (Rothstein and Reznikoff 198 1).

Derivatives of Tn5, with direct rather than inverted terminal repeats of IS50,

have been generated and found to transpose about as well as Tn5 wild type. They

are somewhat unstable in recombination proficient host cells, however, because ho-

mologous recombination between the IS elements results in the loss of one IS50

element plus TnS’s central region. In addition, the central segment of Tn5 can be

replaced by other DNA segments without impairing transposition. Thus we view Tn5

as a composite element, selected because of its antibiotic resistance and its trans-

posability. Its mobility arises simply from the ability of pairs of IS elements to move

in unison, carrying with them interstitial segments; the inverted orientation of IS

elements in many transposons could be fortuitous or a reflection of selection for

stability in the face of homologous recombination.

The Ends of IS50

Typically, the base sequence at one end of an IS element is a short, imperfect,

inverted repeat of the sequence at the other end, for example, 15/ 16 bp in IS5,

18/23 bp in ISI, and 36/37 bp in y6 (Iida et al. 1983). Because specific sequences of

such lengths are unlikely to occur in the bacterial genome by chance alone, these

repeats are usually equated with transposase recognition sites, and the sequence mis-

matches in them suggest that transposition does not involve direct base pairing between

IS element ends. (These short terminal inverted repeats within IS elements should

not be confused with the reverse duplication of entire IS elements in transposons

such as Tn5). The ends of IS50 are intriguing because they are only well matched

over eight of the first nine base pairs (fig. 1, bottom). These 9 bp seemed too short

to constitute an entire transposase recognition site, and recent analyses of IS50 mutants

have shown that the recognition sites actually extend 19 bp in from each end, with

only 12 of the

9

positions matched (Johnson and Reznikoff 1983; Sasakawa et al.

1983). Because the outside 19 bp of IS50 seem to be necessary only for transposase

recognition, whereas the 19 bp at the inside end also contain the last few codons of

the transposase gene, the dissimilarity of these two ends may reflect an evolutionary

compromise between the needs for an effective transposase and for an efficient

transposase binding site.

Tests such as those diagrammed in figure 2 had shown that ISSo’s outside and

inside ends differ in activity. Transposition mediated by a pair of outside ends is lOO-

to 1 OOO-foldmore frequent than inverse transposition mediated by a pair of inside

ends. In addition, transposition mediated by one inside end plus one outside end

occurred with an efficiency similar to that mediated by a pair of outside ends. These

results suggested a model in which the c&acting transposase is activated by binding

to the outside end and then tracks along the DNA molecule until it binds a second

outside or inside end with less discrimination (Isberg and Syvanen 198 1; Sasakawa

and Berg 1982).


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PRODUCTS

,.._. _

YIIILU

IO00 I

,----------

_ - - _ _ _ _ __ -.

A[

\

I

I

L_____ _________________________+‘_.’

0 I I 000 IkI

0

DONOR

‘c--

-’

0 0 0 0 1

t

29t3,1_2.9-

--“,

x :

I

I

4 a

’ -_______ _ ________ __ _____ ___--_--++___d

Tronsposition

k-

to A

I I

0

______

__---__.

\

0 0 0

Cl;

12.9 -3.1 A

\

I

\

._______________________________+L.’

IO,0 Ikl 000 1

D -

v

t_3.1-2.9-3.1A ?

,

.________________________________,~_~_~’

3/710

34 /60

26 /60

O/60

FIG. 2.-Functional test of the transposition proficiency of ISWs outside (0) and inside (I) ends.

Shown at left is the 12-kb dimeric pBR333::Tn5 plasmid marked with resistances to ampicillin (a) and

kanamycin (k). IS50 elements are indicated by jagged lines, and the h target sequences are indicated by

dashed lines. The relative positions of the 2.9- and 3.1-kb fragments generated by digestion with XhoI were

used to deduce the structures of the Ran’ Amp’ trans&xition products (Sasakawa and Berg 1982).

Mechanism of Tn5 Transposition

Transposition mediated by IS50 always involves a fragment of the donor DNA

molecule. This suggested that ISSO-mediated transposition is conservative (fig. 3). On

this model, double-strand cleavages separate the mobile DNA segment from its vector,

the element is ligated to its target DNA, and the remainder of the donor DNA

molecule is released as a linear DNA fragment and lost, probably through exonu-

cleolytic degradation. Precocious replication of one of the donor’s siblings fills the

niche created by destruction of the donor molecule, so that in future generations the

mobile element is found at the original site as well as the new location, just as if it

had replicated while (instead of after) transposing (Berg 1977, 1983).

Transposition of other unrelated elements (e.g., Tn3, ISI, and temperate phage

Mu) often produces co-integrate molecules in which the donor and target DNAs are

joined together by direct repeats of the mobile element (See Heffron 1983; Iida et al.

1983; Toussaint and Resibois 1983). The co-integrate structure is generally interpreted

to indicate replicative transposition, and one popular model for how such transposition

might occur is drawn in figure 4.

Until recently there had been widespread acceptance of the notion that all elements

were like Tn3, replicating during transposition. But the findings that Tn5 probably

transposes conservatively, and that phage Mu and ISI can transpose without replicating

(Liebhart, Ghelardini, and Paolozzi 1982; Akroyd and Symonds 1983; Biel and Berg

1984), indicate that, consistent with the known diversity of transposable elements,

there may be a variety of transposition mechanisms, some replicative and some non-

replicative.

Regulation of Transposition

The increase in mobile element copy number associated with conservative as

well as replicative transposition to new sites contributes to the fixation of these elements


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Transposon Tn5 4 15

1

nsertion

I

epair synthesis

loss of donor

FIG.3.-A model of conservative transposition Berg 1977, 1983). The junctions between transposable

element and vector sequences undergo a blunt-end double-strand cleavage and the target DNA molecule

undergoes a staggered double-strand cleavage, the element being inserted between the broken ends of target

DNA, and ligated in place. The ends of the vector DNA are not religated because, unlike the element itself,

they do not contain sites which would be held together by transposase. Hence, a viable circular) product

of Tn5 excision is not formed during transposition. The resultant casting off of the remains of the donor

molecule creates an empty niche which is refilled by precocious replication of a sibling molecule present

in the same cell the new DNA synthesis is indicated by the interrupted lines at bottom).

in bacterial populations. Balanced against this, however, are detrimental effects: each

transposition event is potentially mutagenic, copies of any DNA segment scattered

in the genome facilitate rearrangements via homologous recombination, and excessive

expression of transposable element genes may be inherently deleterious. Tn5 (IS50)

and several other elements are now known to control the frequency of their own

transposition and thereby their proliferation within a genome. The gene in ISSOR,

which encodes transposase, also encodes a second protein, inhibitor, that diminishes

the transposition of IS50 and Tn5.

The inhibitor, unlike transposase, acts in trans, probably interferes directly with

transposase action, and appears to reduce transposition in proportion to the copy

number of ISSOR (Biek and Roth 1980; Isberg et al. 1982; Johnson et al. 1982). By

decreasing the chance that an invading IS50 (or Tn5) element will establish itself,

the inhibitor is advantageous for the resident IS50 Furthermore, although at least


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ltegrate

FIG. 4.-A model of replicative transposition (adapted from Arthur and Sherratt [ 19791; Shapiro

[ 19791). At the onset of transposition the target DNA undergoes a double-strand cleavage, one strand at

each end of the mobile element is cleaved, and the free ends of transposable element and target DNAs are

joined together forming replication forks. DNA synthesis proceeds inward from both ends until the element

is fully replicated. The resultant product is a co-integrate in which donor and target DNAs are joined

together by direct repeats of the element. Co-integrates can be slowly resolved by homologous recombination

between the repeats in recA+ bacterial cells. However, many members of the Tn3 family encode a recom-

bination function, resolvase, which mediates resolution by a site-specific crossover event independent of

the host ret function. The Tn.%encoded resolvase operates so efficiently that T&-based co-integrates have

only been isolated using mutants altered in resolvase’s structural gene or at its site of action. However,

resolution is not necessarily associated with the preceding steps, and hence this step is diagrammed using


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Transposon Tn5

417

one copy of IS50 is beneficial (Hart1 et al. 1983), the inhibitor reduces the rate of

accumulation of additional copies of the resident element to deleterious levels.

Tn3 and ISIO, in contrast, limit transposition by inhibiting transposase synthesis:

Tn3 encodes a protein repressor of the transcription of its transposase gene (Heffron

1983). IS10 encodes a small mRNA molecule that interferes with the translation of

its transposase protein and whose synthesis may interfere with the synthesis of the

transposase message (Symons and Kleckner 1983). In addition, DNA molecules con-

taining one copy of Tn3 generally cannot be used again as targets for Tn3 insertion

(Heffron 1983). Thus, there are numerous ways of controlling transposable element

movement, each of which diminishes the probability that a cell lineage containing

one of these elements will be recolonized by a closely related and potentially competing

element.

Evolutionary Models

We will consider how transposable elements may have arisen from preexisting

immobile genes, then proliferated and diverged.

IS50 Element Formation

Evolution is opportunistic, since genes that initially carry out one role are often

recruited for other functions. We imagine that ISSo’s transposase may be the end

product of a phylogeny begun by chance fusions between genes encoding proteins

that bind to, track along, nick, and reseal DNA molecules, proteins with properties

much like present-day repressors, helicases, and topoisomerases. Some of the

transposase recognition sites currently found at the ends of transposable elements

may have evolved from operators to which ancestral repressor proteins had bound.

Therefore, neither the ancestral genes nor the primordial recognition sites need to

have been part of a preexisting mobile element.

The recognition sites at both ends of an IS element could have arisen by a reverse

duplication of the first binding site. However, the available data also suggest models

in which the two ends do not share a common ancestry (fig. 5). For example, an

incipient IS element may have been created by the linking of a transposase (tvlp) gene

and a single recognition site, “0.” The primitive transposase activated by binding

this 0 site would migrate on the DNA molecule, occasionally encounter another

sequence for which it possessed fortuitous affinity, and cause transposition of the

segment bounded by “0” and this second sequence. Natural selection would favor

mutant elements which transpose efficiently, and mutations would accumulate in the

chance binding sites, in the tnp gene, or, following changes in transposase specificity,

in the “0” sequence itself. Because chance binding sites would be rare, the earliest

IS elements may have been quite long, but shorter derivatives could then have arisen

by spontaneous internal deletions drawing the distant end closer to the tnp gene.

IS elements could also become truncated by the occasional binding of transposase

to other sequences closer to the tnp gene. Once a shortened IS element had moved

from its earlier location, the recapture of its previous recognition site would not occur.

Rather, selection for more efficient transposition would speed the evolution of the

new recognition site, of the tnp gene, and of the original 0 recognition site as well.

The current form of IS50, the dissimilarity between its two ends, and the overlap

between essential coding and recognition sequences may reflect a pressure for com-

pactness inherent in the transposition process itself. Some elements (e.g., y6 and

ISI 721; see Heffron [ 19831) are larger and encode, in addition to transposase, a second

enzyme called resolvase which catalyzes site-specific reciprocal crossovers and the


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-“trip”-

_-_

m-s

“0”

1

incipient

-_

yyT..

~.y...J- -

‘0’

I\

primitive

r

I

mm_

-

'tnp'

---+y___

3

'0

1

- tnp r,__

-me

__

-we

0

I

evolved

FIG. 5.-A model to explain the evolution of an IS element from a simple immobile gene complex.

The boxes represent actual or potential transposase binding sites which differ in DNA sequence. “0,” ‘0,’

and 0 represent successive stages in the evolution of the 0 end binding site, and “trip ” ‘tnp ’ and tnp

represent successive forms of the transposase gene, each encoding a protein of somewhat different specificity

(coadapted to its recognition sites). The right fork represents a spontaneous deletion within the IS element,

and the left fork represents a shortening of the IS element by transposition using an alternative fortuitous

recognition site near to or overlapping the end of the

tnp

gene.

breakdown of co-integrates formed during replicative transposition. Although co-

integrate formation and resolution may have made the evolution of elements in the

y6 family more complicated than that of IS50, it is likely that their current forms

reflect the pressure for decreased size equivalent to that envisioned for IS50.

The Origin of Tn5

Since each of the 1,534-bp repeats of Tn5 is itself a mobile (IS50) element, it

is likely that Tn5 was formed by insertions and/or genome rearrangements which

placed a pair of IS50 elements on both sides of a previously immobile (chromosomal)

segment encoding resistances to kanamycin and to streptomycin. Either original in-

sertions of IS50 or a subsequent rearrangement or deletion separated the kan and str

genes from their original promoter. The resulting weak resistance caused by readthrough

transcription from the transposase gene in ISSOL would have conferred a low level

of resistance to kanamycin and related antibiotics and hence would have been ad-

vantageous to bacteria growing in antibiotic-contaminated environments. Transpos-

ability would have helped the resistance determinant proliferate and become fixed

in the genomes of its new bacterial hosts and thus allowed time for the selection of

a better kan promoter. The formation of this promoter probably required only a

single base pair change in ISSOL. The simultaneous inactivation of the

tnp

gene is

unlikely to have impaired mobility, since the mutant allele is recessive to the functional

tnp+

allele present in the linked ISSOR element.

Comparisons of the DNA sequences of Tn5 and of another transposon, Tn903,

indicate relatedness between their kanamycin resistance genes but not their insertion

sequences (Beck et al. 1982). This suggests the repeated incorporation of previously

immobile resistance genes into new transposons. The apparent inability to translate

the message of TnS’s str gene in E.

col i

Putnoky et al. 1983; Selvaraj and Iyer 1984)

suggests that Tn5 did not arise in enteric bacteria. The finding of significant sequence

homology between the kanamycin resistance genes of Tn5 and those from

Streptococcus


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Transposon Tn5 419

(Trieu-Cuot and Courvalin 1983) and from Streptomyces (Thompson and Gray,

1983) provides direct support for the intriguing hypothesis of Benveniste and Davies

( 1973) that the aminoglycoside resistance genes of gram-negative bacteria may have

come from the gram-positive organisms that produce aminoglycoside antibiotics.

Transposons unrelated to Tn5 probably arose by an analogous series of insertions,

genome rearrangements, and deletions which in some cases extended into the IS

element, rendering one (Tnl721) or both (Tn3) IS elements immobile (see Heffron

1983).

Transposable Element Maintenance and Proliferation

A half-dozen different species of IS elements have been found in the genome of

Escherichia coli K- 12 (see Iida et al. 1983), a laboratory strain that was isolated from

nature many years ago. Several IS elements are present at a level of five to 10 copies

per chromosome. It is likely that similar numbers of elements are present in the

genome of many other bacterial clones, and we are now beginning to understand

factors that contribute to the abundance of transposable elements and their proliferation

within a genome; among the factors are (i) proliferation inherent in their mechanisms

of transposition, (ii) favorable mutations they cause, (iii) benefits host cells may derive

directly from expression of their transposition genes, and (iv) their participation in

networks of intergeneric gene exchange.

Doolittle and Sapienza (1980) and Orgel and Crick (1980) proposed that trans-

posable elements constitute a special class of DNA termed “selfish DNA.” Although

transposable elements might increase by chance alone (Ohta and Kimura 198 l),

Doolittle and Sapienza (1980) and Orgel and Crick (1980) maintained that inherent

in the mechanism of transposition is a tendency for the element to increase relative

to other genomic sequences. Two aspects of this concept now require modification.

First, it had been argued that the current abundance of transposable elements is

attributable solely to replication during transposition. Although it is obvious that

replicative transposition increases transposable element copy number (fig. 4), the

converse, that any increase in copy number demonstrates a replicative mechanism,

is not true. As shown in figure 3, during conservative transposition the vector from

which the mobile element had been taken is lost, and this loss is compensated by

the overreplication of a sibling molecule to fill the empty niche. A cell lineage which

retains a copy of the element at the original site as well as a second copy at a new

site results just as surely as if replication had accompanied, rather than followed,

transposition.

Second, Doolittle and Sapienza ( 1980) argued that “no other explanation for

the origin or maintenance of transposable elements is necessary . . . the search for

other explanations [other than transposition] may prove, if not intellectually sterile,

ultimately futile.” Despite the dramatic impact of this statement, such a position is

counterproductive. Given the tendency of evolution to tinker by mutation with preex-

isting genes, thereby recruiting them for new roles often quite different from those

they had originally fulfilled, it seems naive to imagine that transposable element genes

never confer a selective advantage on their hosts. Recent studies have shown that the

presence of ISSOR or Tn5 permits entire populations of

E. coli

cells to adapt more

rapidly to sudden environmental shifts in chemostat cultures (Biel and Hart1 1983;

Hart1 et al. 1983). The increased fitness of the ISSO-containing population is evident

immediately rather than after a long lag, is independent of the position of IS50 or


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Tn5 in the genome, and is also independent of the movement of IS50 to new sites.

Thus IS50 seems to make its contribution through generally improved cell physiology

rather than an increased frequency of favorable mutations. This intriguing contribution

to fitness, probably mediated by ISSOR’s transposase or inhibitor function, may have

preceded the formation of IS50 as a mobile element.

A second contribution that transposable elements can make to bacterial fitness,

the occasional induction of favorable mutations, has been illustrated by experiments

with TnlO (Chao et al. 1983). The advantage that TnlO confers, although real, appears

less rapidly than that conferred by Tn5, probably because TnlO-induced favorable

mutations occur relatively rarely and long periods of time are required for cells

containing these mutations to outgrow those with the parental genotypes. Because

IS50 makes a direct physiological contribution to all cells in a population, the occasional

beneficial mutations which it must also induce have not been evident.

The ability of IS elements to mediate the transposition of auxiliary genes also

increases the numbers of bacteria harboring these elements. For example, once Tn5

had formed, the frequency of TnS-containing bacteria increased in many environments

simply through the preferential killing of Kan” organisms that lacked Tn5 and re-

population of the niches by clones that contained Tn5. The increased frequency of

Tn5 would increase the sizes of the reservoirs from which solo IS50 elements could

transpose.

Divergence

As transposable elements increase in copy number, they are subject to selective

forces in addition to those operating on immobile genes. Transposition, whether

conservative or replicative, causes proliferation of the mobile elements and thus places

each member of a family in competition with its siblings resident in the same cell.

Under these conditions elements encoding transposases that act preferentially in cis

and inhibitors that act in trans would be favored. Such transposase and inhibitor

proteins should partially isolate each IS element from its siblings in the same cell

and thereby permit the gradual divergence of the members of the family. In cases of

transposase and inhibitor competing for the same sites at the ends of an element, the

divergence of members of a transposable element family would be selected directly.

Acknowledgments

We are grateful to D. Hart1 for stimulating discussions and critical readings of

this manuscript. This work was supported by U.S. Public Health Service Research

grants ROlAI18980 and ROlAI14267 to D. E. B. and ROlAI19919 to

C. M. B.

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MASATOSHI NEI, reviewing editor

Received July 7, 1983; revision received January 23, 1984.

bacterial transposon tn5: evolutionary inference8

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