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Journal of Bacteriology, September 2001, p. 5445-5448, Vol. 183, No. 18
Estonian Biocentre and Institute of Molecular
and Cell Biology, Tartu University, 51010 Tartu, Estonia
Received 9 April 2001/Accepted 11 June 2001
Transpositional activity of mobile elements can be induced by
different environmental stresses. Here, we present evidence that
transposition of Tn4652 is elevated in stationary-phase
Pseudomonas putida and suppressed in an isogenic
Transposons are widespread in
genomes and have important roles in evolution. Transpositional activity
of a mobile element is generally maintained at a low level, yet a high
frequency of transposition may occur in response to certain
environmental stimuli. It has been shown that different stresses, such
as carbon starvation (17), temperature effects (16,
21), and UV light (7), can enhance transposition of
bacterial mobile elements. Moreover, it is hypothesized that activation
of transposition under stress conditions might serve as an adaptive
response to overcome stress and permit new traits to evolve (4,
24). However, the exact molecular mechanisms that underlie
stress-induced transposition remain undefined.
Transposon Tn4652 is a 17-kb-long deletion derivative of the
toluene degradation transposon Tn4651. Pseudomonas putida
strain PaW85 harbors Tn4652 in the chromosome. Mutational
processes in P. putida PaW85 have been previously
studied in starving conditions on phenol minimal plates
(13). That work showed the emergence of phenol-utilizing
mutants due to the activation of transcription of plasmid-encoded
promoterless phenol degradation genes pheBA in the plasmid
pEST1414. About one-third of the starvation-induced Phe+ mutants appeared as a result of insertion of
Tn4652 in front of the phenol monooxygenase gene
pheA (13) (Fig.
1A). The transposition resulted in the
formation of a fusion promoter between the transposon-inverted repeat
and target DNA (13, 19). Interestingly, transposition of
Tn4652 seemed to depend upon the physiological state of
bacteria: transposition frequency increased with time of starvation,
whereas no Tn4652-linked Phe+ mutants
were detected among growing cells of P. putida
(13). This indicated that starvation might increase
transposition activity of Tn4652.
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.18.5445-5448.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Involvement of
S in
Starvation-Induced Transposition of Pseudomonas putida
Transposon Tn4652
ABSTRACT
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Abstract
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S-defective strain. We demonstrate that transcription
from the Tn4652 transposase promoter is controlled by
the stationary-phase-specific sigma factor S. To our
knowledge, this is the first example of direct
stationary-phase-specific regulation of a mobile element transposase.
Data presented in this report support the idea that activation of
transposition under stressful conditions could be an inducible process.
TEXT
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FIG. 1.
(A) Schematic presentation of transposition target
region in plasmid pEST1332. Catechol 1,2-dioxygenase
(pheB) and phenol monooxygenase (pheA)
genes are indicated by grey boxes. Vector DNA of pAYC32 is depicted
with a line. Different insertion sites of Tn4652 are
indicated with arrows. (B) Accumulation of Phe+ mutants on
phenol minimal plates is indicated for P. putida strain
PaW85 (wt) and isogenic rpoS-defective strain PKS54
(rpoS) containing plasmid pEST1332. Each point represents the mean of
five independent determinations, and error bars represent standard
deviations. Dashed lines indicate the theoretical appearance of
Tn4652-linked Phe+ mutants deduced from the
results of PCR analysis of Phe+ colonies. Up to 30 Phe+ mutants were subjected to analysis on each day. (C)
Viability of P. putida PaW85 (wt) and PKS54 (rpoS)
carrying plasmid pAYC32 on phenol minimal plates. Each point represents
the mean of five independent measurements, and error bars represent
standard deviations. 1,0E + 08, e.g., marks 108 viable
cells.
By the adaptation of bacteria to limited nutrient availability, changes
in gene regulation take place, i.e., several genes are shut down while
others are induced. One of the upregulated genes, rpoS,
codes for an alternative sigma factor, S,
which controls expression of multiple stationary-phase genes (10,
18). In order to examine the potential role of
S in the regulation of Tn4652, we
measured transposition of Tn4652 in the wild-type P. putida PaW85 and in an isogenic
S-defective strain.
Transposition of Tn4652 is decreased in the
P. putida rpoS mutant strain.
Transposition of
native Tn4652 was examined in a starvation assay as
described previously (13), except that target plasmid pEST1332 was used instead of pEST1414. Similar to pEST1414, plasmid pEST1332 (15) contains the promoterless pheBA
operon. However, it is more suitable for probing transposition of
Tn4652 since most of the Phe+ clones
arising on phenol minimal plates emerge from the insertion of
Tn4652 (19). Plasmid pEST1332 contains a
specific target site that is preferred over the other sites present in
both pEST1332 and pEST1414. To study the effects of
S on transposition of Tn4652,
plasmid pEST1332 was introduced into P. putida PaW85 and
into its rpoS-defective derivative PKS54. Bacteria were
grown overnight (ON) in Luria-Bertani medium at 30°C and washed with
M9 solution. Approximately 109 cells of ON
cultures of PaW85 and PKS54 were spread onto five phenol minimal
plates, and the accumulation of mutant Phe+
colonies was monitored upon incubation of the plates at 30°C for 7 days. Results presented in Fig. 1B demonstrate that the emergence of
Phe+ mutants in the rpoS-defective
P. putida was strongly suppressed. Appearance of
Phe+ mutants in the rpoS-defective
strain was reduced 5 to 10 times compared to that in the wild-type
P. putida. In order to test the insertions of transposon
Tn4652 into pEST1332, Phe+ mutants
were analyzed by PCR with oligonucleotides pheA, TnR, and TnL (Table
1). PCR analysis of
Phe+ colonies of the P. putida
wild-type strain revealed that more than 95% of these mutants
contained a Tn4652 insertion upstream of the pheA
coding region. In contrast, only about 20 to 30% of the
Phe+ colonies that appeared in the P. putida S-defective strain carried a
Tn4652 insertion in pEST1332 (Fig. 1B). Thus, the absence of
S protein decreased transposition
substantially, by more than 1 order of magnitude, but did not prevent
it entirely. Here, we want to point out that the
Phe+ colonies revealed similar patterns of
insertions in both the wild-type and rpoS-defective strains.
Also, previous results indicate that RpoS is not obligatory for
transcription from the fusion promoters created by Tn4652
insertions (20).
|
S-defective strain decreases
slowly during 14 days of starvation on phenol plates; by the end of the
second week, the number of viable cells of PKS54(pAYC32) had decreased
by 2 orders of magnitude. However, during the first 6 days of
starvation, survival of the S-defective strain
dropped only twofold. This cannot explain how Tn4652-linked
Phe+ mutants had an accumulation rate in
PKS54(pEST1332) that was more than 10-fold lower than that in
PaW85(pEST1332) (Fig. 1B). Therefore, we conclude that
S can act as a positive regulator in
transposition of Tn4652.
Expression of transposase of Tn4652 is
S dependent.
How can RpoS control transposition of
Tn4652? Transposition is mostly regulated by the amount and
activity of transposase, the protein that performs the transposition
reaction. Therefore, we evaluated the amount of transposase (TnpA) of
Tn4652 in a S-defective background
by Western blot analysis with an anti-TnpA polyclonal antiserum. TnpA
is downregulated by the Tn4652-encoded TnpC, and therefore,
the concentration of TnpA in the Tn4652 background is not
detectable by Western blot analysis (12). Yet, TnpA
protein can be shown by this method if the copy number of the
tnpA gene is increased by cloning the tnpA into
plasmid pKT240 [plasmid pKTtnpA(D/H)] (12). Thus, we
performed Western blot analysis with cell lysates prepared from ON
cultures of P. putida PaW85 and PKS54 carrying plasmid
pKTtnpA(D/H). We found that expression of plasmid-encoded TnpA was
substantially decreased in the S-defective
strain; no TnpA protein could be detected by Western blot analysis in
PKS54 (Fig. 2).
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Transcription from the transposase tnpA promoter of
Tn4652 is growth phase controlled and S
dependent.
In order to test whether the Tn4652-encoded
transposase could be under the control of S,
the transcriptional activity of the tnpA promoter (Fig.
3A) was examined in P. putida
strains PaW85 and PKS54. Previously, transcriptional fusions of the
tnpA promoter region with the reporter gene lacZ
have been constructed, and it has been demonstrated that the
tnpA promoter is positively affected by integration host factor (IHF) (11). It has been shown that
S is involved in the regulation of the
expression of IHF in Escherichia coli (1).
Therefore, the tnpA promoter constructs either containing or
lacking the IHF binding site (plasmids pKTlacZS/C and pKTlacZD/C, respectively) were tested in the S-defective
background. The results presented in Fig. 3B demonstrate that the
transcription from the tnpA promoter is entirely dependent on the growth phase of the bacteria. Both reporter plasmids, pKTlacZS/C and pKTlacZD/C, tested in PaW85 exhibited stationary-phase-specific induction of the tnpA promoter. Also, as demonstrated
previously (11), an about five- to sixfold-higher positive
effect became apparent in the presence of the IHF binding site upstream
of the tnpA promoter (Fig. 3B). Measurement of the
-galactosidase expression in the
S-defective P. putida strain PKS54
revealed that no obvious increase could be detected with either
pKTlacZS/C or pKTlacZD/C during growth (Fig. 3B). Bacteria harboring
either plasmid pKTlacZS/C or pKTlacZD/C showed similar and only
slightly detectable levels of -galactosidase activity that remained
50- or 10-fold lower, respectively, than that estimated in the
wild-type strain, and no positive effects of the IHF binding site could
be detected. Thus, these data indicate that stationary-phase-specific
activation of the tnpA promoter specifically requires
S.
|
S- and
70-dependent promoters are generally quite
similar, some subtle but essential differences in promoter sequences
exist to ensure the selectivity between these two major sigma factors.
S-dependent promoters harbor mostly the
sequence CTATACT in the conserved 
10 region
(8), while 70 preferentially
recognizes promoters with the sequence TATAAT. The 10
region CTATGCT of the tnpA promoter of
Tn4652 contains the sequence determinants suggested to be
important for S-dependent transcription, the C
nucleotide upstream of the 10 hexamer and the C at the fifth position
in the 10 hexamer (Fig. 3A). Therefore, we suppose that RpoS
recognizes the tnpA promoter and is directly involved in the
stationary-phase-specific expression of TnpA.
Up to now the role of S in regulation of
transposition has been studied only in experiments with the mutant
bacteriophage Mu. It has been shown that carbon starvation conditions
trigger induction of mutant Mu prophage, resulting in formation of the araB-lacZ coding sequence fusions
(17). Appearance of the araB-lacZ fusion clones on lactose-selective plates was completely abolished in a
S-negative E. coli strain
(9). Since the transposase promoter of Mu was demonstrated
to be not under the direct control of S, it
was supposed that S could regulate Mu
activation indirectly (17). Thus, according to our
knowledge, S-dependent upregulation of the
transposase of Tn4652 is the first example of direct
stationary-phase-specific regulation of a mobile element transposase.
It is well known that plenty of mutations and other types of genetic
variation are associated with the activity of mobile elements.
Transpositional activity of most mobile elements is greatly suppressed,
yet there are several examples of transposons that are activated under
the conditions in which fast genetic changes are needed, i.e., under
different stresses (5, 14, 23). An interesting question
arises: does the activation occur due to malfunction of host defense
mechanisms or is this an induced process to promote mutations that may
potentially contribute to survival in unfavorable conditions? According
to the results presented in this report, we prefer the latter version.
Our results demonstrate that transposition of Tn4652 is
regulated by physiological conditions of the host. In starving
bacteria, transposition of Tn4652 is elevated due to direct
control of the stationary-phase sigma factor S
that is induced just for better survival of cells in stressed conditions. Therefore, we believe that Tn4652 serves as a
good example of transposons that are activated under stressful
conditions to increase the overall mutation rate and to generate new
and potentially useful mutations.
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ACKNOWLEDGMENTS |
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We thank T. Alamäe and A. Tamm for critically reading the manuscript.
This work was supported by grants 4481 and 4482 from the Estonian Science Foundation and by grant no. HHMI 55000316 from the Howard Hughes Medical Institute International Research Scholars Program.
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FOOTNOTES |
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* Corresponding author. Mailing address: Estonian Biocentre and Institute of Molecular and Cell Biology, Tartu University, 23 Riia Street, 51010 Tartu, Estonia. Phone: 372-7-375015. Fax: 372-7-420286. E-mail: rhorak{at}ebc.ee.
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Journal of Bacteriology, September 2001, p. 5445-5448, Vol. 183, No. 18
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.18.5445-5448.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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