Genetic organization of insertion element IS2 based on a revised nucleotide sequence

291

Gelze. 59 (1987) 291-296 Elsevier GEN 02182

Genetic organization (Transposable

element;

of insertion element IS2 based on a revised nucleotide sequence ISI-flanked

transposon;

overlapping

gene)

Hans-J&g Ronecker and Bodo Rak Institut fiir Siologie III, Universitiit, D-7800 Received

17 August

1987

Accepted

24 August

1987

Freiburg [F. R. G.}

SUMMARY

We identi~ed a tr~sposable element resident in the chromosome of ~~c~~~c~~~ coli K-12 strain HB 101. This is an approx. 4400-bp-long transposon flanked by two copies of insertion sequence (IS) I element in direct orientation. One of the ISI elements was found to be integrated into an IS2 element between IS2 bp 139 and bp 140 with the large moiety of IS2 within the transposon. The sequence of this part of IS2 differs from the published sequence of g&M’-308 : : IS2 at a number of positions. Restriction analysis of the published allele, however, indicated that both alleles may in fact be identical. Since six of the eight differences found alter open reading frames, the revised sequence presents a new outlook for the potential genetic organization of IS2.

TNTRODUCTION

Although quite a number of bacterial insertion elements have been characterized on the level of nucleotide sequence, for most of them only very limited information on genetic organization is available (see review by Iida et al., 1983). One reason for this is the fact that the genes of these elements are not Correspondence fo: Dr. B. Rak, Universitlt,

Sch%nzlestrasse

Institut

1, D-7800

fiir

Biologie

Freiburg

III,

(F.R.G.)

Tel. (761)203-2729. Abbreviations: sequence; reading

0378-l

aa, amino acid(s);

kb, kilobase frame;

Il4/87/$03.50

pair(s);

::, novel joint.

0

1987

bp, base pair(s); nt, nucleotide(s);

IS, insertion ORF,

open

correlated with readiiy recognizable phenotypes. Another difficulty may lie in the exceptionally compact organization of some of these elements. We have compared the coding potential of a number of insertion elements and found that, in addition to a larger ORF, often one or two ORFs of significant length are localized on the opposite strand (Rak and von Reutern, 1984). These ORFs are partially or fully contained within the large ORF. In the case of insertion element IS5 we introduced molecular evidence for the presence of one large gene covering almost the entire length of the element and two smaller genes which lie, together with their own promoters, within the large gene on the opposite strand of the DNA (Rak et al., 1982; Rak and von

Elsevier Science Publishers B.V. (Biomedical Division)

292

Reutern,

1984). The products

of all three genes of

~XPERIMENTAI-

IS5 are involved in the process of transposition (F. Brombacher, IS. Fuchs, and B.R., in preparation).

(a) Isolation of the transposon

Due to the exceptionally compact genetic organization of some mobile genetic elements an uneyuivotally defined nucleotide successful genetics. During

analysis

sequence is a prerequisite

using

the selection

the tools

for mutants

a transposable

element

control

region of the bgl operon.

strains of E. cali are unable to grow on as the sole carbon source. Spontaneous

Wild-type &glucosides

for

of molecular

mutations

activating

on acryl-~-glucosides, thereby revealing the presence of a cryptic operon called bgl (Schaefler and

the

cryptic bgl operon of E. coli (Schnetz et al., 1987) in strain HBlOl we isolated Bgl’ mutants which carried

AND RESULTS

integrated

Malamy,

arise, which enable these strains to grow

1969). About 98”/; of all mutants

into the

phenotype (Schnetz et al., 1987). We transformed strain HBlOl (Boyer and Roulland-Dussoix, 1969) with plasmid pFDX733 carrying the wild-type bgl operon (Schnetz et al., 1987), selected for Bgl+ mutants and analyzed the plasmid structure as described previously (Schnetz et al., 1987). Among 875 independent Bgl + isolates two were found to carry an additional segment of DNA of about 4.4 kb within the bgf control region. Restriction analysis reveaIed that in both mutants transposition of the

This transposon,

which is flanked by copies of ISI in direct orientation, contains IS? sequences in its internal region. We determined the nucleotide sequence of the IS2 moiety and found a number of deviations from the published sequence (Ghosal et al., 1979). These deviations are of major consequence for the coding potential of the element since six of them are bp deletions or additions which alter the pattern of ORFs.

I

I

I

I

1

0

1

2

3

4

Fig. 1. Restriction are marked {inverted Schnetz (Messing,

map and organization

repeats)

are marked

Sequencing

to be sequenced

1983) after tilling-in protruding

and the strategy

designations

was done according

were first subcloned

to Maxam

into the SmaI (Klenow,

The sequence

and Gilbert

large fragment).

(1980) modified

restriction

In this manner, The arrows

contiguously

sites

elements

as described

of vector plasmid polylinker

the extent of the individual

of the fragments.

was determined

IS2. Relevant

kbP

The termini of the insertion

site of the polylinker

map represent

specify the relative orientation analysis.

for sequencing

map has not been determined.

The lines above the restriction

and extent of the sequence

were read for all parts.

transposon

restriction

ends with DNA polymerase

and recutting.

which follow the plasmid

mark the direction sequences

by triangles.

et al. (1987). Fragments

could be used for end-labelling numerals

of a @-activating

with black dots in cases where a complete

have an

ISI or IS.5 element integrated within the control region of the operon resulting in the inducible Bgl +

sequences

subclones;

below the restriction

for both strands

by

pUC12 roman map

and overlapping

293

same segment of DNA had occurred. restriction Fig. 1.

analysis

of this transposon

(b) Interpretation

of

the

evidence

Details of the

map

lated from strain HBlOl 2). Enzyme

The restriction map of the inserted DNA suggested that the transposon is flanked by two copies

restriction

data

suggested

membranes Iabelled 715-bp

(Schleicher

with [a-“PIATP

fragment

Plasmid

-

7242

-

-

6369

-

6369

-

-

5686

-

5686

-

-

4822

-

4822

-

-

4324

-

4324

-

3675

-

2323

-

1929

-

& Schuell, Dasselt, as described

IS2-internal

HkzdIII-Hpal

within the control

of the standards

1929

are indicated.

-

-

DNA and chromosomal

from the transposon

-

-

1242

with PslI. The resulting

derived

transposon

8454

8454

-

analysis.

2

-

2323

F.R.G.),

by Feinberg

DNA were prepared

fragments

were separated

and hybridized and Vogelstein

(panel A), an appron. fragment

labelled (Fig. 1): (i)

c

-

-

a

approx. 185-bp SfpI-SspI fragment from the middle part of the transposon (Fig. 2, panel B); (iii) a 7 15bp

ZL

2:

to completion

and radioactively

B

3,

and produced

of 3.6 kb. Three probes

an about 66%bp H&I-SslII fragment from the left arm of the transposon (Fig. 2, panel A); (ii) an

the

A.

Fig. 2. Southern

fragment

were prepared

presence of an incomplete copy of IS2 (Ghosal et al., 1979) within the transposon. To obtain additional

was digested

(Fig. 2, A, B, and C, lanes

PsrI cut once in ISI

transposon-DNA

of ISI (Ohtsubo and Ohtsubo, 1978; Johnsrud, 1979) in direct orientation as diagrammed in Fig. 1. the

and to

DNA of a mutant of plasmid pFDX733 carrying the transposon within the bgl control region (Fig. 2, A, B, and C, lanes 1) and of chromosomal DNA iso-

and

Southern analysis

Furthermore,

of IS2 sequences

learn more about the nature of the transposon we performed Southern analyses with PstI digests of the

are given in

restriction

for the presence

derived

region of the bgl operon;

according

to Southern

(1983). The labelled

185-bp SspI-SspI

from plasmid

by standard

pFDX129

2, chromosomal

techniques

on a 0.7:~ agarose

(1975). DNA fragments probes

fragment

derived

(panel

C). Lanes:

DNA prepared

(Maniatis

et al., 1982). DNA

gel, transferred

to nitrocellulose

used as probes were

were an approx.

665-bp

from the transposon 1, plasmid

from strain

HBIOl.

pFDX733 Positions

Hpal-SsrII

(panel B), and a carrying

the

and sizes (bp)

294

HindIII-&a1 fragment internal to IS2 (Ghosal et al., 1979; Fig. 2 panel C). This fragment was excised from plasmid pFDX129, which carries IS2

(d) Restriction

on a EglII DNA fragment from phage A mutant r32, an insertion mutant with IS2 at the i, bp position

sites revealed

38 350 (Daniels

et al., 1983). Plasmid pFDX129

had

been constructed by inserting the Bg/II fragment, which spans the insertion site, into the singular BgiII

A computer

analysis search for the presence five discrepancies

lished IS2 sequence newly determined quence recognition

(Ghosal

of restriction

between

the pub-

et al., 1979) and the

sequence: In the published sesites for NluIV, FokI, SfuN and

site of vector plasmid pFD.5 1 (Rak and von Rcutern,

Nael are present at IS2 bp 265, bp 897, bp 919, and bp I 13 1, respectively. All of these are absent in our

1984). All three probes hybridized

sequence.

fragment

to the 3.6kb

excised from a plasmid-borne

PstI

copy of the

recognition

On the other site at position

hand,

we found

an NciI

1132, which is absent in

transposon as well as to a chromosomal DNA fragment of identical size indicating that the DNA

the previously published sequence. digested DNA preparations of

inserted into the plasmid is present colinearly in the chromosome. Moreover, hybridization with the IS2internal probe (panel C) verified the presence of IS2 elements in the transposon. Hybridization with the

308: :IS2 (plasmid pDGl2 of Ghosai et al., 1979) and of the transposon-associated IS2 with enzymes FokI, NueI, and Neil (not shown). The analysis clearly showed that in both alleles FokI and NueI did not cut at the critical position, whereas NciI did. Restriction patterns of both alleles were identical. These findings are strong indication that there are errors in the published sequence of IS2 allele 308 and that this allele may be identical to that found in the transposon.

IS2-probe also indicated the presence of at least eleven copies of IS2 within the chromosome of strain HB 101. Probe (i) gave a single signal with the chromosomal DNA, suggesting that this sequence only occurs within the transposon. Probe (ii), on the other hand, gave two signals (panel B), indicating that part of the internal structure of the transposon was present in at least two copies in the chromosome one of which was definitely not located in the transposon. (c) Sequence of the IS2 moiety Starting at a BstNI-site on the right-hand copy of ISI of the transposon (ISI bp 654, Ohtsubo and Ohtsubo, 1978; Johnsrud, 1979) we determined the nucleotide sequence of some 1450 bp to the left. The sequence obtained verified the presence of IS1 on the ~ght-hand side of the transposon. ImmediateIy adjacent to the terminus of ISI (not shown) the sequence obtained corresponded to IS2 sequences starting at IS2 bp 140. The sequencing strategy is given in Fig. 1. The nucleotide sequence determined for the portion of IS2 present in the transposon is shown in Fig. 3. For comp~ison, the sequence of IS2 allele 308 (Ghosal et al., 1979) is also given. We found eight discrepancies: 2 nt exchanges (marked by asterisks) and six single nt deletions or additions (marked by solid triangles). Potential translational start and stop codons for ORFs are also given for both sequences.

We therefore allele galUP-

(e) Conclusions An approx. 4.4kb ISl-flanked transposon is present in the genome of E. coli K- 12 strain HB 10 1. Southern analysis showed that part of the internal region of the transposon is present only in transposon-form in the chromosome of strain HB 10 1. A second segment is present in a context distinct from that of the transposon in at least one copy. One copy of IS2 (IS2 bp 140-1331) was found inside the transposon and immediately adjacent to one of the flanking ISI elements. The nucleotide sequence of this part of IS2 differs in eight positions from the published sequence of IS2 (allele 308 in Ghosal et al., 1979; Fig. 3). A comparative restriction analysis of the two alleles indicated that both sequences are identical at the three critical positions examined and conform to the sequence presented here. A fourth deviation, an additional C residue at bp 1220, has previously been noted for allele 308 by Rak and von Reutern (1984) and for two other alleles by Ahmed et al. (1980) and by Brosius and Walz (1982). The first 139 bp of IS.2 not present in our transposon have been independently verified by Ogata et al. (1982) and the first 127 bp by Brosius

295

1

-&stop ORFZ TGGATTTGCCCCTATATTTCCAGACATCTGTTATCACTTAACCCATTACAAGCCCGCTGCCGCAGATATTCCCGTGGCGAG s.top

ORFl

border

of IS2

in

tranwoson

1.GCTTCTTTGCTGCCGTTAACCCGTCTGGTTTGGGCATGATACTGATGTAGTCACGCTTTAT . . . . GGTTCTTTGCTGCCGTTAACCCGTCTGGTTTGGGCATGATACTGATGTAGTCACGCT~AT

.

101

GCGGATGCCATTCGTTATAATGCTCGAACGCCTCTGCAA

aoi 201

* . CGTTTTCACGMGCTCTCTGCTATTCCGTTACTCTCCGGACTCCGCACCGCCGTGTTCTTCGGTTCAAGTCCCAACATCCGGGCGAACTGGCGTGTTTCA CGTTTTCACGAAGCTCTCTGCTATTCCGTTACTCTCCGGACTCCGCACCGCCGTGTTCTTCGGTTCCAGTCCCAACATCCGGGCGAACTGGCGTGTTTCA

301 301

TTAGCCCGGTAGCATGAACCATTATCCGTCAGCCACTCCACTGGAGACGACGGAAGATCGTTGCCGAAGCGGCGTTCCACCGCTCCCAGCATGACGTCCT TTAGCCCGGTAGCATGAACCATTATCCGTCAGCCACTCCACTGGAGACGACGGAAGATCGTTGCCGAAGCGGCGTTCCACCGCTCCCAGCATGACGTCCT

401 401

GTACTGTTTCACTGTTGAAGCCGCCGGTAGTGACCGCCCAGTGCAGTGCCTCACGATCACAGCAGTCCAGCGCGAACGTGACACGCAGTCTCTCTCCGTT CTACTGTTTCACTGTTGAAGCCGCCGGTAGTGACCGCCCAGTGCAGTGCCTCACGATCACAGCAGTCCAGCGCGAACGTGACACGCAGTCTCTCTCCGTT

501 501

ATCACAGCAGAACTCGAACCCGTCAGAGCACCATCGCTGATTGCTTTCTTTCACGGCCACTCTGCCTGTATGTGCCCGTTTCGATGGCGGTACAGCAGGT ATCACAGCAGAACTCGAACCCGTCAGAGCACCATCGCTGATTGCTTTCTTTCACGGCCACTCTGCCTGTATGTGCCCGTTTCGATGGCGGTACAGCAGGT

601 601

TTTCGCTCAAGCAACAGCGCATTCTGGCGCATGATCCGGTAAACACGTTTGGCATTGATCGCAGGCATACCATCAAGTTCTGCCTGTCTGCGMGCAGCG TTTCGCTCAAGCAACAGCGCATTCTGGCGCATGATCCGGTAAACACGTTTGGCATTGATCGCAGGCATACCATCAAGTTCTGCCTGTCTGCGAAGCAGCG

701 701

CCCATACCCGACGATAACCATACGTTGGCAGCTCTCCGATAACATGGTGTATACGGAGAAGCACATCCGTATCATCAGTGTGACGACTGCGGCGGCCATC

801 801

CATCCAGTCATCGGTTCGTCTGAGAATGACGTGCAACTGCGCACGCGACACCCGGAGACAACGGCTGACTAAGCTTACTCCCCATCCCCGGGCAATAAGG CATCCAGTCATCGGTTCGTCTGAGAATGACGTGCAACTGCGCACGCGACACCCGGAGACAACGGCTGACTAAGCTTACTCCCCATCCCCGGGCAATAAGG

t

start

ORF4.s

stlr‘t 0RF3

-&Start

start

0RF3

CCCATACCCGACGATAACCATACGTTGGCAGCTCTCCGATAACATGGTGTATACGGAGAAGCACATCCGTATCATCAGTGTGACGACTGCGGCGGCCATC

s

901 901

stop

G

=

E

ORFZ

start

start

0RF3

stop

0RF2

ORF4 y~$

stop

SstoP

UAA -

0RF3

GCGCGTGCGCTATCCACTTTTTTGCCCGTCCATATTCAACGGCTTCTTT~AGGAGTTCA~TTTCCATCTGTTTTCTTGCCGAGCAGGCGCTGGAGTTCTT GCGCGTGCGCIATCCACTTTTTTGCCCGTCCATATTCAACGGCTTCTTTGAGGAGTTCATTTTCCATCAGTTTTCTTGCCGAGCAGGCGCTGGAGTTCTT ORF4

-

G”G

sstxt

.

ORFl

1000 1000

TMTCTGCTTCATGGCGGCAGCAAGTTCAGAGGCAGGAACAACCTGTTCTCCGGCGGCGACAGCAGTAAGACTTCCTTCCTGGTATTGCTTACGCCAGAG TAATCTGCTTCATGGCGGCAGCAAGTTCAGAGGCAGGAACAACCTGTTCTCCGGCGGCGACAGC~GTAAGACTTCCTTCCTGGTATTGCTTACGCCAGAG

1100 1099

AAATAACTGGCTGGCTGCTACACCATGTTGCCGGGCAACGAGGGAGACCGTCATCCCCGGTTCAAAGCTCTGCTGAACAATTGCGATCTTTTCCTGTGTG AAATAACTGGCTGGCTGCTACACCATGTTGCCGGICAACGAGGGAGACCGTCATCCCCGGTTCAA~GCTCTCCTGAACAATTGCGATCTTTTCCTGTGTG

1200 1097

GTACGCCGTCTGCGTTTCTCCGGCCCTAAGACATCAATCATCTGTTCTCCAATGACTAGTCTAAAAACTAGTATTAAGACTATCACTTATTTAAGTGATA GTACGCCGTCTGCGTTTCTCCGGCCATAAGACATCAATCATCTGTTCTCCAATGACTAGTCTAAAAACTAGTATTAAGACTATCACTTATTTAAGTGATA

1300 la97

TTGGTTGTCiGGAGATTCAtGGGGCCAGTiTA TTCGTTGTCTGGAGATTCAGGGGGCCAGTCTA

sstart

Fig. 3. Nucleotide

sequence

of IS2. The sequence

published

IS2 sequence

previously

published

translation

start codons

sequences.

Arrows mark the direction

above the sequence; marked

ORFl

by a vertical

(lower lines). The presently

sequence

(Ghosal

ORFl

in this study (upper lines) is aligned with that of the previously

of IS2 as determined determined

sequence

starts at IS2 bp 140. Nucleotides

et al., 1979) as verified by other authors

(AUG and GUG) followed by ORFs of significant

base-pair

of the reading frames. Base-pair

deletions

are represented

by blackened

(Ogata

et al., 1982; Brosius

length with the respective

exchanges triangles.

and Walz, 1982). Potential

stop codons

between the two sequences The border

1 to 139 are taken from the are indicated

are marked

of the IS2 sequence

for both

with an asterisk

in the transposon

is

arrow.

and Walz (1982). We compiled the complete sequence of IS2. According to this sequence IS2 is 133 1 bp long, 4 bp longer than previously reported (Ghosal et al., 1979). In Fig. 4, the ORFs deduced from the previous sequence are compared with those deduced from our compiled sequence. There are obvious differences: we find four ORFs of significant

length (Fig. 4A). ORFl, 2 and 3 lie on one strand of the DNA and could encode proteins of 121 aa (M, 13 452), 30 1 aa (M, 34 366), and 97 aa (A4, 11 115), respectively. A fourth ORF, located on the opposite strand, could code for a protein of 143 aa (M, 16415).

296

Fig. 4. Schematic orientation Ghosal

representation

of ORFs starting

of the potential

coding capability

of 152 as deduced

with either GTG or ATG. (A) Sequence

as determined

from ORFs. The arrows in this communication.

give the direction

(B) Sequence

and

taken from

et al. (1979).

Johnsrud,

ACKNOWLEDGEMENTS

L.: DNA sequence

Mol. Gen. Genet.

We thank H. Saedler for plasmid pDG 12. Edward Schwartz and Christoph Beck critically read and improved the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft through grants

Ra276/3-6

Mania&T.,

Fritsch,

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with base-specific

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chemical

cleavage.

end-labeled

Methods

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(1980) 499-560.

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J.: New Ml3 vectors

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