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Journal of Bacteriology, December 2001, p. 6943-6946, Vol. 183, No. 23
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.23.6943-6946.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
IS186 Insertion at a Hot Spot in the
lon Promoter as a Basis for Lon Protease Deficiency of
Escherichia coli B: Identification of a Consensus
Target Sequence for IS186 Transposition
L.
SaiSree,1
Manjula
Reddy,1 and
J.
Gowrishankar1,2,*
Centre for Cellular and Molecular Biology,
Hyderabad 500007,1 and Centre for
DNA Fingerprinting and Diagnostics, Hyderabad
500076,2 India
Received 2 July 2001/Accepted 5 September 2001
 |
ABSTRACT |
The radiation sensitivity of Escherichia coli B was
first described more than 50 years ago, and the genetic locus
responsible for the trait was subsequently identified as
lon (encoding Lon protease). We now show that both
E. coli B and the first reported E. coli
K-12 lon mutant, AB1899, carry IS186
insertions in opposite orientations at a single site in the
lon promoter region and that this site represents a
natural hot spot for transposition of the insertion sequence (IS)
element. Our analysis of deposited sequence data for a number of other
IS186 insertion sites permitted the deductions that (i)
the consensus target site sequence for IS186 transposition is
5'-(G)
4(N)3-6(C)4-3', (ii) the associated host sequence duplication varies within the range of 6 to 12 bp and encompasses the N(3-6) sequence, and (iii) in a
majority of instances, at least one end of the duplication is at the
G-N (or N-C) junction. IS186-related sequences were absent in closely related bacterium Salmonella enterica
serovar Typhimurium, indicating that this IS element is a recent
acquisition in the evolutionary history of E. coli.
| |
TEXT |
The heritability of radiation
sensitivity and radiation resistance traits in bacteria was first
studied by Evelyn Witkin more than 50 years ago (26, 27),
at a time when the genetic basis of inheritance in these organisms was
still a matter of debate (14, 26, 27). Her choice of
Escherichia coli B as the strain for these studies was
historically significant, not least because it is the most radiation
sensitive of all natural isolates of E. coli
(14). In 1964, Howard-Flanders et al. (12)
reported isolation of the first E. coli K-12 lon
mutant, AB1899, and stated that "as regards sensitivity to radiation,
... K-12 lon
resemble strains
B... " That E. coli B (as well as its
radiation-resistant suppressor derivative E. coli B/r
[26, 27]) is also a natural lon mutant was
shown subsequently by Donch and Greenberg (9). Deficiency
of the Lon protease (encoded by lon) in the B strains is one
reason that they have been developed as E. coli hosts of choice for overproduction of recombinant heterologous proteins (2, 24, 28).
We now report that the lon mutations in both E. coli B and AB1899 (E. coli K-12) were caused by
IS186 insertions in opposite orientations at a single site
within the spacer region of the lon promoter and that this
site is a natural hot spot for IS186 transposition. An
unusual feature of this insertion sequence (IS) element is that, unlike
most other transposons (7, 25), it is associated with a
variable length of duplication of the flanking host sequence at the
sites of its insertion (5, 23).
Independent lon::IS186
insertions at a single site in four strains.
We showed earlier
(21) that K-12 strain GJ1823 had suffered a spontaneous
and unselected IS186 insertion at the same site in the
spacer region of the lon promoter as that identified by Ignatov and Chistyakova (13) on a plasmid derivative
(pBLI) with the cloned lon gene. The genotype description
for strain AB1899 at the E. coli Genetic Stock Center
website (http://cgsc.biology.yale.edu) indicates, as a personal
communication from D. A. Vlazny and C. W. Hill, that the
lon-1 mutation in the strain is caused by IS186 insertion although its position is not specified.
These facts provided us the rationale to compare, at the molecular
level, the features of the lon mutation in strains AB1899, B/r, and GJ1823 with that published earlier for the plasmid pBLI by
Ignatov and Chistyakova (13). We initially performed PCRs using pairwise combinations of the following three primers, designated A, B, and C, respectively: 5'-TGACCAAGCAGTATCAGG-3',
5'-AAGATCGTTTACACCCGGCT-3', and
5'-GAGAAGCTGATTATATCGT-3'. Primers A and B were designed to amplify in PCR the region from 
392 to +277 of lon
(relative to the start site of lon transcription, taken as
+1). Primer C is complementary to a site within the 1.34-kb
IS186 element, situated 0.6 kb away from one of its ends.
With primer pair A and B, the PCR product obtained with genomic DNA of
wild-type E. coli strain MG1655 as the template was 1.3 kb
smaller than that obtained with genomic DNA from each of the
lon mutants AB1899, GJ1823, and B/r (Fig.
1). With the DNA templates from the three
lon mutant strains, primer pair A and C on the one hand and
pair B and C on the other behaved mutually exclusively, in that a PCR
amplification product was obtained only with the latter for strains
AB1899 and GJ1823 and only with the former for strain B/r (Fig. 1).
From these findings and from the calculated sizes of the PCR products,
one could conclude that all three strains have suffered
IS186 insertion mutations in the vicinity of the
lon promoter (data not shown) and that the orientation of
IS186 in strains AB1899 and GJ1823 was opposite to that in
E. coli B/r.
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FIG. 1.
PCR analysis of lon locus in various
E. coli strains. Primers A and B (corresponding to two
sites in lon) and primer C (corresponding to an internal
site in IS186) were used for PCR in the pairwise
combinations indicated with genomic DNA templates of the following
strains: 1, MG1655 (K-12 wild type); 2, GJ1823 (K-12
lon); 3, AB1899 (K-12 lon); 4, B/r
(lon). Methods for PCR and electrophoresis on 0.9%
agarose gel were as described previously (22). At the left
are shown the positions of DNA markers (sizes in kilobases). Estimated
sizes of the observed PCR products were as follows: MG1655 (primers A
and B), 0.7 kb; all lon mutants (primers A and B), 2.0 kb; AB1899 and GJ1823 (primers B and C), 1.0 kb; E. coli
B/r (primers A and C), 1.1 kb.
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|
DNA sequence analysis of the PCR products obtained above allowed the
precise determination of both the site of
lon::IS
186 insertion and the extent of
the flanking target sequence duplication
for strains AB1899, GJ1823,
and B/r. These data are compared in
Fig.
2 with the findings for the
lon::IS
186 mutation on plasmid
pBLI
reported earlier (
13). The results indicate that, although
all four insertions occurred at a single site in the spacer region
of
the
lon promoter (given that the sequences reading from the
left to the start of IS
186 in all of them are identical),
each
insertion is molecularly distinct from the other three in terms
of
IS
186 orientation or length of target sequence duplication
or both. The inferred pair of sites of staggered endonucleolytic
cleavage of the
lon target sequence during the process of
IS
186 transposition in each of the four instances are
depicted in Fig.
2.
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FIG. 2.
Molecular identities of various
lon:: IS186 insertions. Shown
on top is the (double-stranded) sequence of the relevant region of the
wild-type lon+ locus (4),
and the 10 motif in its promoter region is overlined. Beneath are
given (lines 1 through 4) the lon locus sequences
determined in this study (using an automated DNA sequencer and
associated protocols) for K-12 strains AB1899 and GJ1823 and for
E. coli B/r, as well as that reported earlier
(13) for plasmid pBLI. In each case, the
IS186 sequence (uppercase) is denoted in an abbreviated
form by a few nucleotides from the inverted repeat at either end and by
the orientation of IS insertion (marked, respectively, as I or II
depending on whether the PstI site in
IS186 is to the left or to the right of the
BamHI site within the element); the orientation in pBLI
(*) was not reported (13). The IS186
sequence itself is represented as having a 25-bp pair of inverted
repeats as its ends (15). The flanking host sequence
duplications are boxed. Arrows, postulated pairs of staggered
endonucleolytic cleavage sites (1 through 4, as above) in the target to
generate the four different IS186 insertions.
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|
Copy number of IS186 in different strains.
Whole-genome sequence analysis of wild-type E. coli K-12
strain MG1655 has shown that there are three copies of IS186
in its chromosome (4) (Table
1), and a previous Southern blot
hybridization study came to the same conclusion (15). The
latter study also reported that E. coli B has the same
IS186 copy number as K-12. Our present results (that
E. coli B is a lon::IS186
mutant) therefore raised the possibility that IS186
transposition into lon may have occurred by a cut-and-paste
mechanism (7) from another chromosomal site in the B
strain, followed by the rejoining (that is, recircularization) of the
two ends of the broken donor backbone at the latter site. However, such
rejoining is considered to be very rare in bacteria (7).
To address this issue further, we examined the chromosomal
IS
186 copy number in MG1655, K-12
lon::IS
186 mutants AB1899 and
GJ1823,
and
E. coli B derivatives B/r and BL21 (
24) by
Southern
blot hybridization. A radiolabeled
BamHI-
PstI fragment of IS
186 was used
as probe for hybridization with genomic DNA preparations
of the various
strains after digestion with enzyme
EcoRV,
BglI,
or
PvuII, none of which cuts within the IS
186
element. The results,
shown in Fig.
3,
clearly demonstrate that MG1655 has three copies
of IS
186
and that K-12
lon::IS
186 derivatives
AB1899 and GJ1823
each have an additional copy which correlates with
the insertion
in
lon. The two
E. coli B
derivatives each have five copies of
IS
186, the most
plausible explanation for which is that they represent
insertions in
the loci homologous to the three of wild-type K-12,
with
additional copies in
lon and near
hokX
(
16). In another
study, Birkenbihl and Vielmetter
(
3) also concluded that an
IS
186 insertion into
a new locus in a K-12 strain represented
an additional (fourth) copy of
the IS element.
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FIG. 3.
Southern blot hybridization of genomic-DNA preparations
from various strains using the IS186 probe. Experimental
methods were as described previously (22). The DNA
preparations were digested with EcoRV,
BglI, or PvuII as indicated and subjected
to Southern blot hybridization with a 32P-labeled
PstI-BamHI internal fragment of
IS186 (obtained from plasmid pHYD138
[21]), followed by autoradiography. Strains employed: 1, MG1655 (K-12 wild type); 2, B/r; 3, AB1899 (K-12 lon);
4, GJ1823 (K-12 lon); 5, BL21 (E. coli
B); 6, S. enterica LT2. At the left are shown the
positions of migration of DNA markers (sizes in kilobases). Arrows,
lon-specific bands in the lanes corresponding to AB1899
and GJ1823; their sizes (5.6 [EcoRV] and 8.4 kb
[BglI]) are consistent with those expected from
IS186 insertion in the cognate
lon-bearing restriction fragments of wild-type K-12
(4, 20).
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|
In the Southern blot hybridization experiment (Fig.
3), we also
included bacterial genomic DNA from strain LT2 of closely
related
organism
Salmonella enterica serovar Typhimurium and
observed
no signal for the IS
186 probe. Our finding is
consistent with
the fact that IS
186 sequences have not so
far been detected in
the unfinished genome sequence of
S. enterica (
http://www.tigr.org).
IS
186 has also not been
detected in the genome of
E. coli O157:H57
(
10,
17) or in any of the other sequenced bacterial genomes,
indicating that this IS element, much like others such as
IS
2,
IS
4, and IS
30 (
8),
may be a recent acquisition in the evolutionary
history of
E. coli. The findings of Pedersen and Gerdes (
16)
suggest that IS
186 is present in
E. coli C and
also in not less
than 44 of the 72 natural
E. coli isolates
of the ECOR
collection.
Consensus sequence for IS186 transposition.
Different IS elements are known to vary in the extent to which they
exhibit target site selectivity for transposition (7, 25).
A consensus sequence for IS186 transposition has not
previously been determined, although it is known that the insertions
occur in G-C rich target sequences (5, 15, 23, 25).
Because many eukaryotic sequences cloned in E. coli hosts
are G-C rich, several instances of IS186 insertions in them
are identified in the GenBank database. We analyzed all the deposited
entries to deduce that sequence
5'-(G)4(N)3-6(C)4-3'
is the consensus target recognition site for IS186
transposition (Table 1). The lon hot spot fits the
consensus, as do all but one of the entries listed in Table 1. Our
analysis also indicates that, even as its length varies between
6 and 12 bp, the associated host sequence duplication always
encompasses the (N)3-6 sequence of the target
recognition site. Furthermore, in more than half of the instances
examined at least one end of the duplication (that is, at least one
site of endonucleolytic cleavage during transposition) is at the G-N
(or N-C) junction (Table 1).
These findings may offer approaches to study the mechanism of
IS
186 transposition, of which at present little is known.
Our
analysis suggests that, in IS
186 transposition, there is
an initial
specificity in target sequence recognition by transposase
but
that the sites where subsequent endonucleolytic cleavages occur
are
less constrained; such a phenomenon invites comparisons with
both the
mechanism of type I restriction endonuclease action (
18)
and that of rearrangements of the immunoglobulin genes mediated
by the
RAG1 and RAG2 proteins, where a preference for G-C-rich
sequences at the target sites (
11) and the generation of
duplications
of variable length (
1) have also been
observed.
It has been suggested that IS
186 belongs to the
IS
4 family of insertion elements (
5,
15,
19,
25), but the family
as currently defined includes members that
generate variable (IS
4 and IS
186) as well as
fixed lengths of target site duplications.
The length of the
duplications associated with transposition of
IS
50
(Tn
5), which is another member of the IS
4 family,
is usually
9 bp but rarely could also be 10 bp (
6).
Finally, IS
1 is also
known to generate host sequence
duplications of various lengths
(
25) but is not a member
of the IS
4 family.
| |
ACKNOWLEDGMENTS |
We thank Mary Berlyn for bacterial strains and acknowledge the
assistance of Mehar Sultana and N. Nagesh with oligonucleotide synthesis and DNA sequencing, respectively. We also thank one of the
anonymous reviewers for suggesting the analogy between IS186 transposition on the one hand and restriction
endonuclease action and antibody gene rearrangement on the other.
J. G. is an honorary faculty member of the Jawaharlal Nehru Centre
for Advanced Scientific Research.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centre for DNA
Fingerprinting and Diagnostics, ECIL Rd., Nacharam, Hyderabad 500076, India. Phone: 91-40-7155609. Fax: 91-40-7155610. E-mail:
shankar{at}www.cdfd.org.in.
| |
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Journal of Bacteriology, December 2001, p. 6943-6946, Vol. 183, No. 23
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.23.6943-6946.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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