Mutagenesis, Vol. 14, No. 1, 129-134,
January 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Effect of Tn10/Tn5 transposons on the survival and mutation frequency of halogen light-irradiated AB1157 Escherichia coli K-12
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5A, 02-106 Warszawa, Poland
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Abstract |
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We show here that the Tn10/Tn5 transposon when inserted into the chromosome of strain AB1157 makes the bacteria more sensitive to and less mutable by halogen light irradiation. These effects are most probably caused by depletion of UmuD and UmuC proteins since: (i) transformation of the transposon-bearing bacteria with plasmids harbouring umuD'C (or umuDC) leads to recovery of the original survival and mutation frequencies; (ii) insertion of Tn10/Tn5 into chromosomal DNA has no effect on the level of mutation induced by ethyl methanesulphonate treatment, a mutagen whose activity is umuDC-independent; (iii) the decline in survival is in about the same range for Tn10-bearing bacteria as for bacteria with deleted umuDC. However, whereas transformation of bacteria deleted in umuDC with plasmids carrying umuD'C/umuDC leads to full recovery of halogen light-induced mutability, recovery of survival is poor. This suggests that the mechanisms leading to umuDC-dependent mutagenesis and umuDC-dependent protection of cell survival are different. None of these effects occurs in bacteria bearing the Tn9 transposon in their DNA.
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Introduction |
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Halogen light emits a broad spectrum of UV radiation (UVA, UVB and UVC) and is a strong mutagenic agent whose mechanism and specificity of mutations are similar to those of UV254 nm irradiation and results from the formation of pyrimidine dimers and 6-4 pyrimidine photoproducts in DNA (De Flora et al., 1990
; Wójcik and Janion, 1997; Fabisiewicz and Janion, 1998). Reversion of argE3(ochre) to Arg+ in Escherichia coli strain AB1157 after halogen light irradiation occurs predominantly either by de novo supB suppressor formation or by supEamber to supEochre suppressor conversion; both mutations occur in tRNA genes by transition of GC to AT base pairs at the appropriate sites in the genomic DNA (Wójcik and Janion, 1997). Formation of supB, but not of supE(ochre), leads to the MFD phenomenon, a decline in mutant frequency when bacteria are transiently incubated under non-growing conditions immediately after UV or halogen light irradiation (Witkin, 1956; Doudney and Haas, 1958; Bridges et al., 1967; Bockrath and Palmer, 1977; Wójcik and Janion, 1997, Fabisiewicz and Janion, 1998). This is the result of preferential repair of the transcribed strand of DNA (Selby et al., 1991; Selby and Sancar, 1994). The T-C sequence, a target for UV attack in glnU (the source of supB), is situated on the transcribed strand, and in supE44 (the source of supE(ochre)) on the non-transcribed (coding) strand.
Transposons are widely used as tools for construction of bacterial strains and as carriers for genetic material. It has been found, however, that zcf117::Tn10 is not an indifferent insertion in bacterial cells but makes bacteria proficient (UvrA+) and deficient (UvrA–) in nucleotide excision repair more sensitive to and less mutable by halogen light and UV irradiation (Wójcik and Janion, 1997; Fabisiewicz and Janion, 1998). These effects are studied here in a greater detail using bacteria with Tn10 transposons inserted at various sites in DNA and halogen light as the source of UV irradiation. It has been shown here that the decrease in survival and in mutation frequency is not dependent on the site of transposon insertion and that both effects are the result of UmuD and UmuC deficiency, since both of them may be suppressed by overproduction of the UmuD and UmuC proteins. However, whereas in bacteria with deleted umuDC and transformed with a plasmid overproducing UmuDC there is full recovery of halogen light-induced mutagenesis, recovery of survival is poor. This suggests that mechanisms leading to umuDC-dependent mutagenesis and to umuDC-dependent recovery of survival may be different.
Of the investigated transposons only insertion of Tn10 and Tn5 influences the survival and mutagenicity of the irradiated cells, whereas insertion of transposon Tn9 does not.
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Materials and methods |
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Bacterial strains and plasmids
The strains used were E.coli K-12 AB1157 with the relevant genotype argE3(ochre), hisG4(ochre), thrA(amber), supE44-amber suppressor (Bachman, 1987), EC2413 (as AB1157 but
umuDC595::cat; Woodgate, 1992; Grzesiuk and Janion 1994) and their transposon- or plasmid-bearing derivatives. The transposons and plasmids used for construction of bacterial strains are listed in Tables I and II. The names of transduced and transformed bacteria were denoted by adding the name of the insert (or plasmid) to the bacterial strain, e.g. AB1157-zcf117::Tn10 or AB1157/pBR322. Transduction (via P1 phage) and transformation were performed by routine methods (Miller, 1972; Sambrook et al., 1989) and selection was on Bulion (B) or Luria broth (LB) plates containing ampicillin/kanamycin/spectinomycin/chloramphenicol at 50 µg/ml or tetracycline at 15 µg/ml. Plasmids pGW were from the collection of Dr G.C.Walker, pRW134 from Dr R.Woodgate. The bacteria bearing transposons were kindly donated by Drs R.M.Shaaper, R.Devoret, K.Yamamoto, D.Hulanicka and A.Bebenek.
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Media
For growth of bacteria, B or LB (Miller, 1972
) with addition of ampicillin, tetracycline, kanamycin spectinomycin and chloramphenicol, when necessary, was used. Minimal medium (MM) was C-salt (Vogel and Bonner, 1956) with 0.4% glucose. Revertants of Arg+ were selected on Arg– agar minimal plates, enriched with all requirements except Arg: Thr, Leu and Pro at 20 µg/ml and Thi at 10 µg/ml.
Halogen light irradiation
Bacteria grown in LB to ~2–3x108 cells/ml were centrifuged, resuspended in the same volume of MM and irradiated (9 ml in a 90 mm Petri dish) with a halogen lamp (150 W; Polamp) placed 20 cm above the plate. A 1 min irradiation emitted 162 J/m2 UVC, 846 J/m2 UVA and 732 J/m2 UVB, measured using an IL-1500 radiometer (Dexter Industrial, Green Newburyport, MA). The bacteria were diluted in LB (0.3–3 ml), grown overnight, then diluted and, after plating on B and Arg– plates, the viable cells and Arg+ revertants were scored after 2 days incubation. The survival of the irradiated bacteria (percentage of the number before and after irradiation) was estimated. The survival and Arg+ reversion frequency after 5 min halogen light irradiation corresponds to that obtained by UV254 nm irradiation at a dose of 40 J/m2.
All incubations were performed at 37°C and the irradiated bacteria were kept under dim yellow light or in darkness.
Ethyl methanesulphonate (EMS) treatment
Bacteria grown overnight in LB medium (with antibiotic when necessary) were treated with 2% EMS for 40 min at 37°C. After centrifugation and washing, a 30 µl sample was resuspended in 3 ml LB, incubated overnight and plated onto B and Arg– plates. Viable counts and Arg+ revertants were scored after incubation for 1 (viable cells) or 2 days (Arg+ revertants).
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Results |
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Characteristic of the transposon-bearing bacteria
The transposons listed in Table I
were transduced into the AB1157 strain to provide a collection of bacteria bearing transposons at different sites of the genome. The sequences neighbouring all of the transposons (except those arising from transposon insertion) were of the wild-type, showing that no new phenotypes were introduced, except those arising from transposon insertion. The phenotypes arising from transposon insertion are not known for all the transductants. We do not know the sequences of DNA, coding or non-ncoding, at which transposons are inserted at the sites denoted zcf, zbd and x near endA. The latter strain is used in our laboratory to transfer endA mutations after passage of the transposon in an endA– strain. Insertion of the construct leuB::Tn10 leads to the phenotype Leu–, but since strain AB1157 is leuB6, the phenotype of AB1157-leuB::Tn10 remains the same. Insertion of the transposon at sites srlC300 or malB impairs permeability of bacterial membranes or influences their metabolism and, hence, the bacteria are unable to grow on sorbitol or malabiose as a sole source of carbon.
Since the Tn10 transposon sensitizes both UvrA+ and UvrA– excision repair-deficient bacterial cells to UV irradiation and the effects are not dependent on the site of transposon insertion, it can be concluded that none of the transposon insertions have any influence on DNA repair systems.
Effects of Tn10/Tn5 transposons and plasmid pGW2123 on the survival and reversion frequency of halogen light-irradiated AB1157 derivatives
Bacteria bearing a transposon ± plasmid pGW2123 (umuD'C) were irradiated with halogen light and their survival and Arg+ reversion frequency were estimated. The results in Table III clearly show that all of the AB1157 derivatives bearing the Tn10 or Tn5 (but not the Tn9) transposon were much more sensitive to and less mutable by halogen light irradiation than the parental AB1157 transposon-free strain. The effects are moderate, but clearly evident. After 5 min irradiation survival due to the presence of the transposon was decreased ~10-fold and the frequency of mutation by a factor of ~2-fold. The effects remain the same when a tetracycline resistance determinant in the Tn10 transposon was substituted by a kanamycin resistance determinant.
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Since halogen light is a umuDC-dependent mutagen and does not produce mutations in bacteria deficient in umuDC, it was natural to suppose that the reason underlying the effects arising from transposon insertion may be a decline in UmuD(D')C proteins. This hypothesis seems to be correct; transformation of transposon-bearing bacteria with plasmid pGW2123 overproducing UmuD'C suppresses the effects of the Tn10/Tn5 transposon insertion and increases survival and mutation frequency in halogen light-irradiated cells. The survival is almost fully restored and the level of Arg+ revertants is even higher in AB1157-zcf117::Tn10/pGW2123 transformants than in the AB1157 strain (Table III
). For example, the frequency of reversions to Arg+ (per 108 cells) in bacteria irradiated for 5 min, which decreases from 1676 in AB1157 to 471 in AB1157-leuB::Tn10 or to 693 in AB1157-zcf117::Tn10, increases to 2792 and 3451, respectively, after transformation with plasmid pGW2123. However, transformation by pGW2123 of AB1157 lacking Tn10 also leads to an increase in the reversion frequency (from 1676 to 2400) and to a small decrease in survival (from 51 to 31%). A small decrease in survival is also noted in AB1157/pBR322 transformants (see Table IV, compare data in the first three rows).
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These results suggest that: (i) UmuD'C proteins are the limiting factor for mutagenesis induced by halogen light irradiation for both types of bacteria, those having Tn10/Tn5 transposons as well as the transposon-free AB1157 strain; (ii) transformation with plasmids exerts two effects that may counteract each other, one leading to a decrease in survival as a result of transformation with any plasmid and the other leading to an increase in survival as a result of overproduction of plasmid-expressed UmuD'C. Because the Tn10 transposons are inserted at different sites on the genome of AB1157, the resulting bacterial strains are genetically different. Therefore, both effects, sensitivity to halogen light irradiation and lower ability to mutate, result from Tn10/Tn5 transposon insertion and not from sequences accidentally picked up with the transposon from donor strain DNA.
Suppression of Tn10 effects in AB1157-zcf117::Tn10 by transformation with plasmids harbouring umuD, umuD', umuDC or umuD'C genes
To gain more information about the role of UmuD(D')C proteins in survival and mutagenesis of halogen light-irradiated bacteria, the AB1157-zcf117::Tn10 strain was transformed with plasmids carrying either umuD, umuD', umuDC or umuD'C and, after irradiation, the survival and reversion frequencies were determined. The results (Table IV) show that recovery of survival is greatest in transformants overproducing UmuD'C and poorest in transformants overproducing UmuD, whereas recovery of ability to induce mutations is more or less similar in all of these transformants overproducing either UmuD, UmuD', UmuDC or UmuD'C (~3400 Arg+/108 bacterial cells).
Therefore, for halogen light-induced survival, similarly as for mutagenesis, both UmuD(D') as well as UmuC are required and whereas chromosomally expressed UmuC protein is sufficient for mutagenesis to occur, it is not enough for restoration of survival. On the other hand, since umuD (or umuD' alone) is in sufficient amounts to recover mutation frequency, it can be concluded that UmuD protein is a major limiting factor responsible for mutagenicity decline in Tn10-bearing cells.
Note that the increase in halogen light survival and mutagenicity in response to transformation with a plasmid bearing umuD'C genes is in about the same range for pGW2123 (~30 copies/cell) as for pRW134 (3–5 copies/cell).
Effect of overproduction of plasmid-expressed UmuD, UmuD', UmuDC or UmuD'C on survival and mutagenesis of halogen light-irradiated strain EC2413 (like AB1157 but umuDC)
Deletion of umuDC increases the sensitivity of bacteria to UV and halogen light irradiation to a level comparable with that observed in AB1157 (umuDC+) with the Tn10/Tn5 transposon insertion and makes cells totally non-mutable by UV or halogen light. Insertion of Tn10 into umuDC bacteria does not change their sensitivity to halogen light irradiation (Table V). No recovery of survival (or mutation) is seen in bacteria deficient in chromosomal umuDC and transformed with a plasmid harbouring either umuD or umuD' alone. However, transformation with plasmids carrying umuDC or umuD'C restores mutability of halogen light-irradiated cells to the level observed in umuDC+, but survival is poorly restored. The greatest increase in halogen light survival was from 1.3% for the umuDC-deleted strain to 9% in its pGW2123 transformant, whereas in AB1157-zcf117::Tn10 (umuDC+) the greatest increase after pGW2123 transformation was 27%. Note that transformation of umuDC bacteria (EC2413 zcf117::Tn10) with plasmids overproducing UmuDC (pGW2101) or UmuD'C (pGW2123) does not suffice for restoration of Arg+ reversion frequency to the level of that observed in pGW2101/pGW2123 transformants of the EC2413 transposon-free strain (Table V).
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Two possible reasons may be advanced for this effect: either the mechanism of protection by UmuDC against decrease in survival differs from that leading to mutations or different lesions are involved in these two processes. It is also possible that the umuDC operon responds to halogen light-induced expression much more rapidly when in the bacterial chromosome than when in the plasmid and that this delay has no consequences for umuDC-dependent mutagenesis, whereas it does for survival.
Effect of Tn10/Tn5 transposon insertion into strain AB1157 on survival and mutagenesis after EMS treatment
EMS is a mutagen whose activity does not depend on umuDC. To verify, whether the effect of the Tn10/Tn5 transposon on survival and mutation frequency is restricted to mutagens whose activity is umuDC-dependent, we treated bacteria with 2% EMS and measured the level of survival and mutations in AB1157 and in its transposon-bearing derivatives (Table VI). The results show that neither survival nor mutation frequency was influenced by Tn10/Tn5 insertion.
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Discussion |
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In this paper it was shown that transposons Tn10 (9.3 bp) and Tn5 (5.7 bp), but not Tn9 (2.6 bp), when inserted into chromosomal DNA, render cells more sensitive to and less mutable by halogen light or UV irradiation. The effects are moderate but should not be neglected, since they increase with an increase in irradiation dose and the acquired phenotypes may be wrongly interpreted as mutations transferred with the help of the Tn10/Tn5 transposons into a newly constructed strain.
Insertion of the transposons has no effect on viability of non-irradiated bacteria nor on the time of restarting of bacterial growth after halogen light irradiation (data not shown).
Both the decline in survival and in mutation frequency are not dependent on the site of transposon insertion and may be suppressed by overproduction of the UmuD(D')C proteins. These results therefore indicate that: (i) the effects result from insertion of the transposon itself and not from other sequences acquired from the donor strain; (ii) in bacteria with the Tn10/Tn5 transposon there is a shortage of UmuD(D')C proteins.
The role of UmuD and UmuC in SOS-induced mutagenesis is well established. The proteolytic function of RecA* induced after an SOS response triggers expression of umuDC as well as the other SOS genes and processing of UmuD to UmuD' (Nohmi et al., 1988; Sweasy et al., 1990; Frank et al., 1993; Walker, 1995; Peat et al., 1996). Mutation occurs as a result of replicative bypass of non-coding bases (pyrimidine dimers, 6-4 pyrimidine photoproducts or apurinic sites) with the help of RecA, UmuD'2UmuC and DNA polymerase III (Bridges and Woodgate, 1985; Rajagopolan et al., 1992; Woodgate and Levine, 1996). The levels of UmuD, UmuD' and UmuC are tightly controlled by transcriptional and post-translational regulation. Overproduction of UmuDC switches RecAmediated recombination to RecA–UmuD'2C–DNA pol III-mediated mutagenesis (Baillone et al., 1991). Overproduction of UmuD'C may greatly inhibit survival of UV-treated bacteria (Sommer et al., 1998), but it should be noted that in Tn10/Tn5 transposon-bearing cells overproduction of UmuDC as well as UmuD'C leads to an increase in survival as well as of mutations (Tables III and IV).
An increase in halogen light-induced mutagenesis (versus AB1157) was also found in AB1157/pGW2123 overproducing UmuD'C (Table III) and in AB1157/pGW2020 overproducing UmuD (data not shown). This suggests that UmuD either has some as yet unknown role in metabolism of DNA in which UmuD(D') is involved or that UmuD(D') plays a critical role in production of mutagenic UmuD'2UmuC proteins.
Measurement of the levels of UmuD and UmuD' proteins by western blot in SOS-induced bacterial strains without and with the Tn10 transposon seems to exclude the straightforward explanation that the transposon lowers expression of the umuDC operon or processing of UmuD to UmuD'. There are no striking differences in the level or processing of UmuD to UmuD' in SOS-induced bacteria transformed with pGW2101(umuDC) with or without the transposon. As to the level of UmuD induction, the sensitivity of the method is too low to detect a possible small decrease in chromosomal umuDC expression in response to insertion of the transposon (E.Speina and C.Janion, unpublished data).
Since the work of Woodgate and Ennis (1992) it is known that the UmuD protein, in contrast to UmuC, is in great excess; there are ~200 molecules of UmuD and 16 of UmuC per non-induced cell, which after SOS induction increase, respectively, to 2800 (930 UmuD + 1900 UmuD') and 200 molecules/cell. The great excess of UmuD over UmuC is therefore maintained after SOS induction.
Surprisingly, we have found that overproduction of UmuD or UmuD' in AB1157-zcf117::Tn10, bacteria proficient for umuDC, was sufficient to recover the mutagenicity (but not survival) which had been decreased in response to transposon insertion. The possibility that in the transposon-bearing strain UmuD (or UmuD') is more available for some reaction which arises as a result of Tn10 insertion is difficult to evaluate. It is known that in SOS-induced cells the abilities to excise and to transpose Tn10/Tn5 transposons are enhanced (Kuan and Tessman, 1992; Levy et al., 1993), but whether this may influence the level of Umu(D)D' is not known.
UmuD interacts with activated RecA–ssDNA and this interaction promotes processing of UmuD to UmuD' (Story et al., 1992). Similarly, UmuD'C associates with and remains bound to single-stranded DNA and to RecA-coated DNA (Frank et al., 1993; Bruck et al., 1996). Therefore, it is reasonable to suppose that the level of UmuD protein available for the processes of mutation may be changed by its sequestration on single-stranded DNA. Perhaps another role of UmuD(D'), in cooperation with RecA, is protection of single-stranded DNA from degradation. We have no explanation as to why chromosomally expressed UmuDC is more potent in maintaining survival of UV-irradiated cells than plasmid expressed UmuDC. The survival of halogen light-irradiated bacteria is clearly higher in AB1157-zcf117::Tn10/pGW2123 than in EC2413 (AB1157 but umuDC)/pGW2123 transformants, whereas the frequencies of Arg+ revertants in these two strains are similar (Tables IV and V). This may reflect a better availability of chromosomal than plasmid UmuDC for chromosomal DNA repair or a less efficient expression or more rapid degradation of plasmid-borne UmuDC. The differential response to overproduction of plasmid UmuDC, which leads to a greater increase in the level of mutagenesis than of survival after halogen light irradiation, may be related to the different mechanisms of these reactions. Mutations occur when a wrong base is inserted opposite a non-coding site, a pyrimidine dimer (or 6-4 photoproduct), without interruption of the DNA strand, whereas repair of irradiated DNA is more complex and requires excision of modified bases and nicking of the DNA and hence may be less effective.
In summary, It is most probable that Tn10/Tn5 transposons influence survival and mutagenicity of UV-irradiated bacteria by depletion of UmuD(D') and UmuC proteins. Although the exact mechanism remains unknown, the potential phenotype of higher UV sensitivity and lower UV mutability must be considered when new bacterial strains are constructed with the help of Tn10/Tn5 transposons.
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Acknowledgments |
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We are greatly indebted to Drs G.C.Walker, R.Woodgate, R.M.Shaaper, R.Devoret, K.Yamamoto, D.Hulanicka and A.Bebenek for bacterial strains and phages and to Dr R.Hancock for friendly language correction. This work was partially supported by the State Committe for Scientific Research, grant no 6 P04A 034 08.
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Notes |
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1 To whom correspondence should be addressed. Tel: +48 658 47 66; Fax: +48 39 12 16 23; Email: celina{at}ibb.waw.pl
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Received on July 8, 1998; accepted on September 16, 1998.
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