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Inhibition of sulfur incorporation to transfer RNA by ultraviolet-A radiation in Escherichia coli
Journal of Photochemistry and Photobiology B: Biology 71 (2003) 69–75 www.elsevier.com/locate/jphotobiol
Inhibition of sulfur incorporation to transfer RNA by ultraviolet-A radiation in Escherichia coli Oscar J. Oppezzo *, Ram on A. Pizarro Departamento de Radiobiologı´a, Comisi on Nacional de Energı´a At omica, Av. General Paz 1499, 1650 General San Martı´n, Buenos Aires, Argentina Received 17 February 2003; received in revised form 1 August 2003; accepted 1 August 2003
Abstract tRNA sulfurtransferase activity was assayed in Escherichia coli cell extracts obtained from bacterial suspensions exposed to a sub-lethal dose of ultraviolet-A radiation (fluence 148 kJ m2 ) imparted at a low fluence rate (41 W m2 ). We found that the irradiation reduced the enzymatic activity to one fourth of the control value, indicating that ultraviolet-A exposure inhibits the synthesis of 4-thiouridine, the most abundant thionucleoside in E. coli tRNA. Changes in the tRNA content of 4-thiouridine and its derived photoproduct 5-(40 -pyrimidin 20 -one) cytosine were studied in bacteria growing under ultraviolet-A irradiation. In these conditions the accumulation of photoproduct was limited, and the kinetics of this process was non-coincident with disappearance of 4-thiouridine. The results, which are compatible with the fact that ultraviolet-A induces an inhibition of the 4-thiouridine synthesis, suggest that the effect of radiation on tRNA modification is relevant to tRNA photo-inactivation in growing bacteria. Ó 2003 Elsevier B.V. All rights reserved. Keywords: Escherichia coli; Ultraviolet A; Growth delay; tRNA; 4-Thiouridine
1. Introduction The modified nucleoside 4-thiouridine (s4 U) is widely distributed in bacterial transfer ribonucleic acid (tRNA). In Escherichia coli, about 70% of the tRNA molecules contain this thionucleoside, which is present almost exclusively at position 8 and strongly absorbs ultraviolet-A radiation (UVA) [1]. In tRNA molecules carrying cytidine in position 13 in addition to s4 U, UVA exposure triggers a photoreaction that results in the formation of an intra-molecular covalent cross-linking between positions 8 and 13, with generation of the photoproduct 5-(40 -pyrimidin 20 -one) cytosine (Pyo(4– 5)Cyt). This modification produces a restriction in the aminoacylation capacity of tRNAPhe and tRNAPro , reducing the rate of protein synthesis and leading to the increase in the guanosine tetraphosphate level dependent on the product of the relA gene. As a consequence of these events, cells of E. coli exposed to UVA undergo *
Corresponding author. Tel.: +54-11-6772-7008; fax: +54-11-67727188. E-mail address: [email protected] (O.J. Oppezzo). 1011-1344/$ - see front matter Ó 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2003.08.004
a transient inhibition of growth without loss of viability, called growth delay [2]. This effect plays a relevant role in bacterial response to UVA [3,4] and contributes to the modification of the bacterial response to ultraviolet C [5]. The events responsible for triggering the growth delay have been described in detail, but some aspects of this phenomenon, such as the complex dependence of the growth inhibition on the fluence rate [6] and the mechanism by which cells resume growth after irradiation [7], remain unclear. Recent studies concerning the UVA effects on Enterobacter cloacae and E. coli showed that, in addition to the cross-linking between s4 U and cytidine residues, the irradiation of bacteria at low temperature induces a transient reduction in the s4 U content of tRNA during post-irradiation growth [8]. This result suggests that UVA could exert an inhibitory effect on s4 U synthesis in bacterial cells exposed to a sub-lethal dose of radiation at a low fluence rate, similar to those expected in natural environments [9]. Since s4 U is responsible for the first event in the mechanism of the growth delay effect, a modification in s4 U content of tRNA during UVA exposure could modify the growth delay induction, and
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any phenomenon related to this effect in bacterial response to radiation. The aim of the present study was to investigate the inhibitory effect of UVA on tRNA thiolation in E. coli, evaluating the eventual influence of such an inhibition on the growth delay induction in physiological conditions. The results obtained, allowed us to propose an explanation for the limited effectiveness of UVA to induce growth delay in growing cultures of E. coli.
2. Materials and methods 2.1. Bacterial strains and growth conditions Escherichia coli K12 (ATCC 15153) was used throughout this study. Salmonella typhimurium DA1915 (cysB403 relA21::Tn10 argC95) was kindly provided by Dr. D. Ant on, Comisi on Nacional de Energıa At omica, Argentina. Luria-Bertani broth (10 g/l tryptone, 5 g/l yeast extract, 8.6 102 M NaCl) [10] was used as rich medium. The E medium of Vogel and Bonner (5.7 102 M K2 HPO4 , 1.7 102 M Na(NH4 )HPO4 , 102 M citric acid, 8.1 104 M MgSO4 ) with glucose 0.5% as a carbon source [11] was used as minimal medium. When required, this medium was supplemented with 5.9 106 M thiamine, 4 104 M nicotinic acid, 9.5 105 M arginine or 1.3 104 M cysteine. Cultures were incubated at 37 °C with shaking and bacterial growth was monitored by measuring the optical density at 650 nm. 2.2. Measurement of tRNA sulfotransferase activity Salmonella typhimurium DA1915 was used as a source of sulfur-deficient tRNA. Bacteria were grown to exponential phase in minimal medium supplemented whit cysteine and arginine, harvested, washed twice with 0.15 M NaCl, and suspended in minimal medium without cysteine [12]. After incubation at 37 °C for 16 h tRNA was obtained from these cells according to a previously described procedure [13]. The content of s4 U in this preparation was 25% of that found in cells grown in the presence of cysteine. Extracts for tRNA sulfurtransferase activity measurements were prepared from E. coli K12 grown to exponential phase in rich medium, suspended in NaCl 0.15 M, and maintained in the dark or exposed to UVA (365 nm, 60 min, 41 W m2 ). The UV source, the dosimetric procedure, and the protocol followed for irradiation, have been described elsewhere [8,13]. Bacterial cells were harvested, suspended in 5 vol of buffer 80 mM tris(hydroxymethyl)-aminomethane hydrochloride, pH 7.8, 8 mM MgCl2 , 1 mM dithiothreitol, 10% glycerol and disrupted by sonication. Extracts were centrifuged at 12 000g for 20 min and the supernatants were further centrifuged at 100 000g for
60 min. Preparations were carried out at 4–8 °C. Sulfurtransferase activity was assayed in the supernatants immediately. In a final volume of 0.5 ml, the reaction mixtures contained: extract, 0.3 ml; sulfur-deficient tRNA, 250 lg; tris(hydroxymethyl)-aminomethane hydrochloride (pH 7.8), 40 lmol; MgCl2 , 4 lmol; ATP, 4 lmol; dithiothreitol, 0.5 lmol; cysteine, 5 nmol; and [35 S]cysteine (>1000 Ci/mmol, Amersham Pharmacia Biotech), 45 lCi. The reaction mixtures were incubated at room temperature during 30 min. The reaction was stopped by the addition of 0.5 ml 88% phenol and, after incubation for 15 min in an ice bath, the mixtures were centrifuged for 30 min at 4 °C and at 20 000g. The top aqueous layers were removed and potassium acetate was added to a final concentration of 2%. tRNA was precipitated from these aqueous solutions by the addition of two volumes of 95% ethanol and centrifugation for 15 min at 4 °C and at 20 000g The resulting precipitates were stored at )20 °C until they were processed. These precipitates were dissolved in 0.6 ml of 0.5 M tris(hydroxymethyl)-aminomethane pH 10 5 mM NaCl and incubated for 90 min at 37 °C to deacylate cysteinyltRNA [12]. After this treatment, tRNA was separated from cysteine and other components of the reaction mixture by gel filtration chromatography on a Sephadex G-100 column (0.9 55 cm) equilibrated and eluted with 0.05 M sodium cacodylate, pH 7, 0.15 M NaCl. Fractions were collected and their absorbance at 260 nm was measured. Aliquots of the fractions corresponding to tRNA (KAV ¼ 0.35) were mixed with scintillation fluid and radioactivity was counted in a TriCarb Liquid Scintillation System (Packard Instruments). The transference of sulfur from cysteine to tRNA during incubation was estimated by the ratio between radioactivity and absorbance in the elution diagrams. Protein concentration in the extracts (2.3 and 1.9 g/l for the irradiated and control extracts, respectively) was measured by the method of Lowry using bovine albumin as a standard. 2.3. Irradiation of exponentially growing bacteria Overnight cultures of E. coli K12 in minimal medium (18 ml) were diluted in 1800 ml of fresh medium previously warmed at 37 °C, and incubated in a rectangular chamber (base, 9.6 cm 47.5 cm; height, 5 cm) provided with a water jacket connected to a constant temperature water bath. The cultures were stirred with magnetic bars to provide aeration. Once the optical density reached 0.15, a bench with two Philips TDL 18W/08 tubes (365 nm) was suspended over the cultures at 1.7 cm from the free surface. The fluence rate at this level was estimated to be 38 W m2 . Alternatively, cultures (1800 ml) were grown at 37 °C with shaking until the optical density reached 0.18, chilled in an ice bath for 15 min, and transferred to the irradiation chamber, which was
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connected to an ice bath. Irradiation conditions were those described above for growing cultures except that the temperature was maintained at 6–8 °C. The optical density of the cultures remained unchanged at a value close to 0.2 during irradiation at low temperature. Immediately after irradiation bacteria were harvested by centrifugation and stored at )20 °C until they were processed for tRNA extraction and analysis. 2.4. Measurements of s4 U and Pyo(4–5)Cyt contents in tRNA The procedures followed for tRNA extraction and hydrolysis, as well as the protocol for reverse phase HPLC analysis of the hydrolysis products has been described previously [13]. The s4 U and Pyo(4–5)Cyt contents were estimated by the areas under the peaks corresponding to these compounds in the chromatograms obtained by HPLC analysis of hydrolysed tRNA. To account for differences in the amount of tRNA present in the samples, the areas under the peaks corresponding to guanosine or guanine were also measured. The results were expressed as the ratio between the areas under the peaks corresponding to s4 U and guanosine, or Pyo(4–5)Cyt and guanine, in the same chromatogram. The values obtained in this way are proportional to the ratio between the concentration of tRNA carrying s4 U, or Pyo(4–5)Cyt, and the total tRNA concentration in each sample. 2.5. Estimation of rate constants for Pyo(4–5)Cyt formation The formation of Pyo(4–5)Cyt follows a pseudo first order kinetics both in vitro [14,15] and in vivo [16–21]. Since in bacteria maintained at 6–8 °C total tRNA concentration remained unchanged, the modifications in the s4 U and Pyo(4–5)Cyt contents during irradiation in this condition depended on photoproduct formation only. In order to calculate the rate constant for the cross-linking reaction at low temperature, a non-linear regression computer program was employed. Using this program we fit the parameters of the following equation to the experimental data obtained during the measurements of s4 U content: cs ¼ co expðktÞ þ cn ;
ð1Þ
where cs is the s4 U content after an irradiation time t, co and cn are the contents of s4 U available and non-available for cross-linking respectively at the start of the reaction, and k is the pseudo first order rate constant. The value of k was also calculated from Pyo(4–5)Cyt accumulation data, fitting the parameters of the following equation to the data obtained: cp ¼ cf ð1 expðktÞÞ;
ð2Þ
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where cp is the Pyo(4–5)Cyt content after an irradiation time t, and cf is the Pyo(4–5)Cyt content after complete reaction. When bacteria were irradiated during the exponential growth phase, measurements of s4 U and Pyo(4–5)Cyt contents were affected by tRNA synthesis and thiolation, in addition to photoproduct formation. As an approximation to analyze the results obtained under these conditions, we assumed that s4 U synthesis stopped quickly when irradiation started and was negligible during UVA exposure. The rate of tRNA accumulation during culture growth is expected to be a function of the rate of stable RNA accumulation [17]. In exponentially growing E. coli irradiated at 366 nm, the effects of radiation on the increase in optical density and on the RNA accumulation were closely similar [18]. In order to take into account the influence of changes in total tRNA concentration on the measurements of s4 U and Pyo(4– 5)Cyt content, a correction, based on the increase in optical density of the culture, was introduced in the calculations. Results obtained with bacteria irradiated at 37 °C were used to fit the parameters of the following equations to experimental data: cs ¼ ðco expðktÞ þ cn Þ=ðODt =ODo Þ;
ð3Þ
cp ¼ ðcf ð1 expðktÞÞÞ=ðODt =ODo Þ;
ð4Þ
where ODt and ODo are the optical densities after an irradiation time t and at the onset of irradiation respectively. The meaning of the other parameters is the same as were described for Eqs. (1) and (2). The optical density expected during irradiation was approximated by the polynomial ODt ¼ 0:149 þ 2:70 104 t 6:26 106 t2 þ 2:33 108 t3 :
ð5Þ
The coefficients included in Eq. (5) were obtained from a typical growth curve by non-linear fitting.
3. Results 3.1. Effect of UVA on tRNA sulfurtransferase activity In order to investigate the effect exerted by UVA on s4 U synthesis, tRNA sulfurtransferase activity was measured in E. coli extracts obtained from irradiated and non-irradiated control cells. When the products obtained after incubation of extracts with sulfur-deficient tRNA, labelled cysteine, and the required cofactors were analyzed, the results shown in Fig. 1 were obtained. Sulfur incorporation found in the extract prepared from irradiated cells was barely one fourth of that observed in the extract obtained from control cells. These results indicate a striking reduction in tRNA sulfurtranferase activity in E. coli after the UVA treat-
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Fig. 1. Sulfur transference from cysteine to tRNA. Sulfur deficient tRNA was incubated with 35 S labelled cysteine, ATP, Mg2þ , and extracts from non-irradiated (panel a) or irradiated (panel b) bacteria. After incubation tRNA was phenol extracted, ethanol precipitated, and deacylated. Finally, tRNA was purified by chromatography on Sephadex G-100. The collected fractions were monitored for absorbance at 260 nm (open symbols) and radioactivity (solid symbols).
ment. Taking into account that 68% of the sulfur contained in S. typhimurium tRNA is incorporated as s4 U [22], a remarkable reduction in the synthesis of this thionucleoside as a consequence of UVA exposure is inferred. 3.2. tRNA modification during growth under UVA irradiation To test whether the effect described in the preceding section could have some influence on the induction of growth delay under physiological conditions, we studied the changes in the tRNA contents of s4 U and Pyo(4– 5)Cyt induced by UVA in exponentially growing E. coli irradiated at a fluence rate similar to that expected in natural environments. The effect of irradiation on the culture growth is shown in Fig. 2. In keeping with data reported for cultures of E. coli exposed to UVA at low fluence rates [6,18], the growth rate decreased without complete cessation of growth, and when irradiation was prolonged beyond 100 min the cultures seemed to grow exponentially but with a longer cell mass doubling time. Radiation effects on tRNA modification are shown in Fig. 3. As a reference, changes in s4 U and Pyo(4–5)Cyt tRNA contents were studied in bacteria irradiated under similar conditions but maintained at low temperature to stop bacterial growth and avoid any effect related with tRNA turnover. As shown in Fig. 3a, in growing cells the reduction in the s4 U content during irradiation was faster than that observed in non-growing cells. The re-
Fig. 2. Effect of UVA irradiation on bacterial growth. Bacteria were incubated in minimal medium at 37 °C. The cultures were maintained in the dark (open circles) or irradiated at 365 nm (solid circles). In the culture exposed to UVA, irradiation started when the optical density reached a value of 0.15 (arrow). Eq. (5) is also plotted.
maining s4 U content in bacteria irradiated at 6–8 °C was always higher than that measured for bacteria incubated at 37 °C and exposed to UVA during the same time. In keeping with several reports on the kinetics of Pyo(4– 5)Cyt formation [14,16], accumulation of Pyo(4–5)Cyt in non-growing cells was concomitant with s4 U disappearance (Figs. 3a and b). After 60 min irradiation at low temperature the photoproduct content was similar to that found when a solution of E. coli tRNA 0.7 mg/ml in sodium cacodylate 0.05 M, pH 7, NaCl 0.1 M was irradiated at 365 nm at a total fluence of 155 kJ m2 . By fitting Eqs. (1) and (2) to experimental data obtained with bacteria maintained at 6–8 °C, the half-times for s4 U content decrease and Pyo(4–5)Cyt accumulation were estimated to be 10.1 and 11.5 min respectively, and 78% of the of s4 U was estimated to be available for cross-linking in this condition. The progress of photoproduct accumulation in growing bacteria seemed remarkably different from that observed at low temperature (Fig. 3b). The Pyo(4–5)Cyt content increased during the first 30 min of UVA exposure, reaching a maximum value corresponding to about 70% of the photoproduct accumulated in nongrowing bacteria after complete reaction. When irradiation was prolonged (for 60 min or more) a reduction in the tRNA Pyo(4–5)Cyt content was observed. By fitting Eqs. (3) and (4) to experimental data obtained at 37 °C, the half-times for s4 U content decrease and Pyo(4–5)Cyt accumulation in this condition were estimated to be 9.9 and 10.2 min respectively, and 71% of the s4 U present in tRNA seemed to be available for cross-linking.
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Fig. 3. Changes in tRNA contents of s4 U and Pyo(4–5)Cyt in bacteria grown under UVA irradiation. Cultures were maintained in the dark (open symbols) or exposed to radiation (solid symbols), while they were growing at 37 °C (circles) or after cooling at 6–8 °C (triangles). At the indicated times bacteria were harvested and tRNA was extracted. The contents of s4 U (panel a) and Pyo(4–5)Cyt (panel b) were measured by reverse phase HPLC analysis of hydrolysed tRNA. Eqs. (1) (panel a, dashed line), (2) (panel b, dashed line), (3) (panel a, solid line), and (4) (panel b, solid line) are also plotted. The values assigned to the parameters involved in these equations were estimated by fitting them to experimental data by non-linear regression. The arbitrary units used in panels a and b are different, by comparing the changes in tRNA contents of s4 U and Pyo(4–5)Cyt after complete reaction in nongrowing conditions it was estimated that 1 A.U. in panel a is equivalent to 0.87 A.U. in panel b.
Since the estimations of the half time for the reaction in both irradiation conditions are similar, and the contents of s4 U and Pyo(4–5)Cyt measured at 37 °C are comparable with the values calculated using Eqs. (3) and (4), the inactivation of s4 U synthesis during irradiation seems to be a suitable approximation to explain our results.
4. Discussion The tRNA-sulfurtransferase activity measurements in UVA-irradiated and non-irradiated bacteria showed that, as we suggested previously [8], the synthesis of s4 U is an UVA-sensitive process. The unique function attributed to s4 U is the triggering of the UVA-induced growth delay, and it is generally accepted that this modified residue acts as a sensor for UVA exposure in bacterial tRNA. In irradiated bacteria, the effectiveness
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of any process related to the growth delay could be conditioned by the ability of UVA to reduce the intracellular concentration of tRNA molecules carrying s4 U. The inhibition of tRNA thiolation is probably a relevant effect in physiological conditions, because it could be induced by low doses of radiation delivered at a low fluence rate. When bacteria are irradiated in non-growing conditions, the cross-linking process involves s4 U synthesized during pre-irradiation growth and the effect of UVA on s4 U synthesis is undetectable. A different situation may be expected when growing cells are exposed to UVA, since in this condition tRNA synthesis and photo-induced cross-linking take place simultaneously, during irradiation. Studying the influence of the dose rate on the induction of growth delay in exponentially growing E. coli cells, Favre [6] proposed a model to relate growth rate and concentration of intact target molecules. According to this model, the extent of the tRNA inactivation depends on the balance between UVA-induced crosslinking of tRNA and de novo synthesis of UVA-sensitive tRNA, so changes in growth rate are explained by the extent of Pyo(4–5)Cyt accumulation in tRNA [6,18]. In our assay conditions, no evident correlation between growth rate and Pyo(4–5)Cyt accumulation was found (Figs. 2 and 3b). Moreover, if during irradiation damaged tRNA were ‘‘diluted’’ with tRNA carrying s4 U, a decrease in Pyo(4–5)Cyt content should be accompanied by a simultaneous increase in s4 U content and, as shown in Fig. 3, this was not the case. The limited accumulation of Pyo(4–5)Cyt and the lack of correlation between variations of s4 U and Pyo(4–5)Cyt contents in growing bacterial cultures exposed to UVA, could be interpreted assuming that UVA inhibits s4 U synthesis in addition to inducing the cross-linking reaction. Experimental data suggest that in growing bacteria exposed to UVA Pyo(4–5)Cyt is formed mainly by reaction of s4 U synthesized before irradiation, and that tRNAs carrying either s4 U or Pyo(4–5)Cyt are ‘‘diluted’’ during irradiation with tRNA lacking s4 U. The tRNA molecules lacking s4 U are biologically active [23,24] but not UVA sensitive. The inhibition of s4 U synthesis should not interfere with protein synthesis, but it would reduce the concentration of the target molecules involved in the first event of the growth delay mechanism. The lack of UVA-sensitive tRNA would limit the accumulation of inactivated tRNA, conditioning the ability of UVA radiation to trigger the growth delay. The concordance between the experimental data and the s4 U and Pyo(4–5)Cyt contents calculated assuming that s4 U synthesis is inhibited during UVA exposure supports this hypothesis. Caldeira de Araujo and Favre [18] reported that in growing cultures of E. coli submitted to UVA at low fluence rate only 80% of the s4 U available for
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cross-linking was converted to Pyo(4–5)Cyt after a prolonged irradiation. Similarly, our calculations estimated that the fraction of s4 U available for cross-linking in growing bacteria was 71% instead of 78% as for nongrowing bacteria. The difference in the availability of s4 U for cross-linking cannot be explained by the inhibition of tRNA thiolation. After 120 min irradiation the content of Pyo(4–5)Cyt decreased faster than expected by dilution of cross-linked tRNA with tRNA synthesized de novo, and the reason for this fact is unknown. An abrupt decrease in the Pyo(4–5)Cyt tRNA content has been described in growing E. coli irradiated at 366 nm with a high fluence rate, and based on this observation the existence of a repair mechanism for crosslinked tRNA was proposed [7]. This repair mechanism has never been demonstrated, but its eventual existence could explain the deviation observed in our experimental data from the expected values at the end of irradiation. On the other hand, growth inhibition described in cultures irradiated at high fluence rates [6] could be produced by quick transformation of previously synthesized s4 U in Pyo(4–5)Cyt. Probably the inactivation of most UVA-sensitive tRNA in a short time could not be compensated by tRNA synthesis. Recent studies performed by Mueller and co-workers [25–27] and by Kambampati and Lauhon [28–30] demonstrated the involvement of the proteins ThiI and IscS in the sulfur transfer from cysteine to s4 U in E. coli. IscS contains pyridoxal phosphate as a cofactor and the absorbance spectrum of the purified protein in its reduced form exhibits a maximum at 363 nm [31]. This characteristic suggests that IscS could be the target for inactivation of s4 U synthesis during irradiation at 365 nm. Both ThiI and IscS are involved in thiamin biosynthesis [30,32] and strains carrying iscS mutations require nicotinic acid in addition to thiamin [30]. To test whether the effect of UVA on the growth rate was related with requirements associated with a deficient activity of IscS, an irradiation assay was performed on a culture growing in minimal medium supplemented with thiamin and nicotinic acid. The effect of UVA on bacteria growing in this supplemented medium was similar to that found in non-supplemented medium (data not shown), and no evidence has been found for the role of IscS in the effect of UVA on s4 U synthesis. It has been proposed that the growth delay protects bacteria against harmful effects of ultraviolet radiation and sunlight [33], but discrepant results were reported when the UVA sensitivity of mutants lacking s4 U was compared with that found in wild type strains. These mutants exhibit increased [34] or reduced [9,35] resistance to the lethal effects of UVA depending on the irradiation conditions. It has been suggested that s4 U is involved in the generation of DNA single strands breaks by UVA [35]. Under certain irradiation conditions, a transient lack of s4 U could help bacteria to avoid dele-
terious effects of UVA related to absorption of radiation by s4 U, increasing survival even when the induction of growth delay and the efficiency of any protective mechanism dependent on this effect is reduced.
Acknowledgements We thank Dr. D.N. Ant on for the bacterial strain ~ez for technical askindly provided, and Mr. J.A. Iban sistance. This research was supported in part by a grant from Fundaci on Balseiro.
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[27] P.M. Palenchar, C.J. Buck, H. Cheng, T.J. Larson, E.G. Mueller, Evidence that ThiI, an enzyme shared between thiamin and 4thiouridine biosynthesis, may be a sulfurtransferase that proceeds through a persulfide intermediate, J. Biol. Chem. 275 (2000) 8283– 8286. [28] R. Kambampati, C.T. Lauhon, IscS is a sulfurtransferase for the in vitro biosynthesis of 4-thiouridine in Escherichia coli tRNA, Biochemistry 38 (1999) 16561–16568. [29] R. Kambampati, C.T. Lauhon, Evidence for the transfer of sulfane sulfur from IscS to ThiI during the in vitro biosynthesis of 4-thiouridine in Escherichia coli tRNA, J. Biol. Chem. 275 (2000) 10727–10730. [30] C.T. Lauhon, R. Kambampati, The iscS gene in Escherichia coli is required for the biosynthesis of 4-thiouridine, thiamin, and NAD, J. Biol. Chem. 275 (2000) 20096–20103. [31] D.H. Flint, Escherichia coli contains a protein that is homologous in function and N-terminal sequence to the protein encoded by the nifS gene of Azotobacter vinelandii and that can participate in the synthesis of the Fe–S cluster of dihydroxy-acid dehydratase, J. Biol. Chem. 271 (1996) 16068– 16074. [32] S.V. Taylor, N.L. Kellerher, C. Kinsland, H.-J. Chiu, C.A. Costello, A.D. Backstrom, F.W. McLafferty, T.P. Begley, Thiamin biosynthesis in Escherichia coli, J. Biol. Chem. 273 (1998) 16555–16560. [33] A. Caldeira de Araujo, A. Favre, Near ultraviolet DNA damage induces the SOS responses in Escherichia coli, EMBO J. 5 (1986) 175–179. [34] G.F. Kramer, J.C. Baker, B.N. Ames, Near-UV stress in Salmonella typhimurium: 4-thiouridine in tRNA, ppGpp, and ApppGpp as components of an adaptive response, J. Bacteriol. 170 (1988) 2344–2351. [35] M.J. Peak, J.G. Peak, L. Nerad, The role of 4-thiouridine in lethal effects and DNA backbone breakage caused by 334 nm ultraviolet light in Escherichia coli, Photochem. Photobiol. 37 (1983) 169– 172.
Inhibition of sulfur incorporation to transfer RNA by ultraviolet-A radiation in Escherichia coli Oscar J. Oppezzo *, Ram on A. Pizarro Departamento de Radiobiologı´a, Comisi on Nacional de Energı´a At omica, Av. General Paz 1499, 1650 General San Martı´n, Buenos Aires, Argentina Received 17 February 2003; received in revised form 1 August 2003; accepted 1 August 2003
Abstract tRNA sulfurtransferase activity was assayed in Escherichia coli cell extracts obtained from bacterial suspensions exposed to a sub-lethal dose of ultraviolet-A radiation (fluence 148 kJ m2 ) imparted at a low fluence rate (41 W m2 ). We found that the irradiation reduced the enzymatic activity to one fourth of the control value, indicating that ultraviolet-A exposure inhibits the synthesis of 4-thiouridine, the most abundant thionucleoside in E. coli tRNA. Changes in the tRNA content of 4-thiouridine and its derived photoproduct 5-(40 -pyrimidin 20 -one) cytosine were studied in bacteria growing under ultraviolet-A irradiation. In these conditions the accumulation of photoproduct was limited, and the kinetics of this process was non-coincident with disappearance of 4-thiouridine. The results, which are compatible with the fact that ultraviolet-A induces an inhibition of the 4-thiouridine synthesis, suggest that the effect of radiation on tRNA modification is relevant to tRNA photo-inactivation in growing bacteria. Ó 2003 Elsevier B.V. All rights reserved. Keywords: Escherichia coli; Ultraviolet A; Growth delay; tRNA; 4-Thiouridine
1. Introduction The modified nucleoside 4-thiouridine (s4 U) is widely distributed in bacterial transfer ribonucleic acid (tRNA). In Escherichia coli, about 70% of the tRNA molecules contain this thionucleoside, which is present almost exclusively at position 8 and strongly absorbs ultraviolet-A radiation (UVA) [1]. In tRNA molecules carrying cytidine in position 13 in addition to s4 U, UVA exposure triggers a photoreaction that results in the formation of an intra-molecular covalent cross-linking between positions 8 and 13, with generation of the photoproduct 5-(40 -pyrimidin 20 -one) cytosine (Pyo(4– 5)Cyt). This modification produces a restriction in the aminoacylation capacity of tRNAPhe and tRNAPro , reducing the rate of protein synthesis and leading to the increase in the guanosine tetraphosphate level dependent on the product of the relA gene. As a consequence of these events, cells of E. coli exposed to UVA undergo *
Corresponding author. Tel.: +54-11-6772-7008; fax: +54-11-67727188. E-mail address: [email protected] (O.J. Oppezzo). 1011-1344/$ - see front matter Ó 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2003.08.004
a transient inhibition of growth without loss of viability, called growth delay [2]. This effect plays a relevant role in bacterial response to UVA [3,4] and contributes to the modification of the bacterial response to ultraviolet C [5]. The events responsible for triggering the growth delay have been described in detail, but some aspects of this phenomenon, such as the complex dependence of the growth inhibition on the fluence rate [6] and the mechanism by which cells resume growth after irradiation [7], remain unclear. Recent studies concerning the UVA effects on Enterobacter cloacae and E. coli showed that, in addition to the cross-linking between s4 U and cytidine residues, the irradiation of bacteria at low temperature induces a transient reduction in the s4 U content of tRNA during post-irradiation growth [8]. This result suggests that UVA could exert an inhibitory effect on s4 U synthesis in bacterial cells exposed to a sub-lethal dose of radiation at a low fluence rate, similar to those expected in natural environments [9]. Since s4 U is responsible for the first event in the mechanism of the growth delay effect, a modification in s4 U content of tRNA during UVA exposure could modify the growth delay induction, and
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any phenomenon related to this effect in bacterial response to radiation. The aim of the present study was to investigate the inhibitory effect of UVA on tRNA thiolation in E. coli, evaluating the eventual influence of such an inhibition on the growth delay induction in physiological conditions. The results obtained, allowed us to propose an explanation for the limited effectiveness of UVA to induce growth delay in growing cultures of E. coli.
2. Materials and methods 2.1. Bacterial strains and growth conditions Escherichia coli K12 (ATCC 15153) was used throughout this study. Salmonella typhimurium DA1915 (cysB403 relA21::Tn10 argC95) was kindly provided by Dr. D. Ant on, Comisi on Nacional de Energıa At omica, Argentina. Luria-Bertani broth (10 g/l tryptone, 5 g/l yeast extract, 8.6 102 M NaCl) [10] was used as rich medium. The E medium of Vogel and Bonner (5.7 102 M K2 HPO4 , 1.7 102 M Na(NH4 )HPO4 , 102 M citric acid, 8.1 104 M MgSO4 ) with glucose 0.5% as a carbon source [11] was used as minimal medium. When required, this medium was supplemented with 5.9 106 M thiamine, 4 104 M nicotinic acid, 9.5 105 M arginine or 1.3 104 M cysteine. Cultures were incubated at 37 °C with shaking and bacterial growth was monitored by measuring the optical density at 650 nm. 2.2. Measurement of tRNA sulfotransferase activity Salmonella typhimurium DA1915 was used as a source of sulfur-deficient tRNA. Bacteria were grown to exponential phase in minimal medium supplemented whit cysteine and arginine, harvested, washed twice with 0.15 M NaCl, and suspended in minimal medium without cysteine [12]. After incubation at 37 °C for 16 h tRNA was obtained from these cells according to a previously described procedure [13]. The content of s4 U in this preparation was 25% of that found in cells grown in the presence of cysteine. Extracts for tRNA sulfurtransferase activity measurements were prepared from E. coli K12 grown to exponential phase in rich medium, suspended in NaCl 0.15 M, and maintained in the dark or exposed to UVA (365 nm, 60 min, 41 W m2 ). The UV source, the dosimetric procedure, and the protocol followed for irradiation, have been described elsewhere [8,13]. Bacterial cells were harvested, suspended in 5 vol of buffer 80 mM tris(hydroxymethyl)-aminomethane hydrochloride, pH 7.8, 8 mM MgCl2 , 1 mM dithiothreitol, 10% glycerol and disrupted by sonication. Extracts were centrifuged at 12 000g for 20 min and the supernatants were further centrifuged at 100 000g for
60 min. Preparations were carried out at 4–8 °C. Sulfurtransferase activity was assayed in the supernatants immediately. In a final volume of 0.5 ml, the reaction mixtures contained: extract, 0.3 ml; sulfur-deficient tRNA, 250 lg; tris(hydroxymethyl)-aminomethane hydrochloride (pH 7.8), 40 lmol; MgCl2 , 4 lmol; ATP, 4 lmol; dithiothreitol, 0.5 lmol; cysteine, 5 nmol; and [35 S]cysteine (>1000 Ci/mmol, Amersham Pharmacia Biotech), 45 lCi. The reaction mixtures were incubated at room temperature during 30 min. The reaction was stopped by the addition of 0.5 ml 88% phenol and, after incubation for 15 min in an ice bath, the mixtures were centrifuged for 30 min at 4 °C and at 20 000g. The top aqueous layers were removed and potassium acetate was added to a final concentration of 2%. tRNA was precipitated from these aqueous solutions by the addition of two volumes of 95% ethanol and centrifugation for 15 min at 4 °C and at 20 000g The resulting precipitates were stored at )20 °C until they were processed. These precipitates were dissolved in 0.6 ml of 0.5 M tris(hydroxymethyl)-aminomethane pH 10 5 mM NaCl and incubated for 90 min at 37 °C to deacylate cysteinyltRNA [12]. After this treatment, tRNA was separated from cysteine and other components of the reaction mixture by gel filtration chromatography on a Sephadex G-100 column (0.9 55 cm) equilibrated and eluted with 0.05 M sodium cacodylate, pH 7, 0.15 M NaCl. Fractions were collected and their absorbance at 260 nm was measured. Aliquots of the fractions corresponding to tRNA (KAV ¼ 0.35) were mixed with scintillation fluid and radioactivity was counted in a TriCarb Liquid Scintillation System (Packard Instruments). The transference of sulfur from cysteine to tRNA during incubation was estimated by the ratio between radioactivity and absorbance in the elution diagrams. Protein concentration in the extracts (2.3 and 1.9 g/l for the irradiated and control extracts, respectively) was measured by the method of Lowry using bovine albumin as a standard. 2.3. Irradiation of exponentially growing bacteria Overnight cultures of E. coli K12 in minimal medium (18 ml) were diluted in 1800 ml of fresh medium previously warmed at 37 °C, and incubated in a rectangular chamber (base, 9.6 cm 47.5 cm; height, 5 cm) provided with a water jacket connected to a constant temperature water bath. The cultures were stirred with magnetic bars to provide aeration. Once the optical density reached 0.15, a bench with two Philips TDL 18W/08 tubes (365 nm) was suspended over the cultures at 1.7 cm from the free surface. The fluence rate at this level was estimated to be 38 W m2 . Alternatively, cultures (1800 ml) were grown at 37 °C with shaking until the optical density reached 0.18, chilled in an ice bath for 15 min, and transferred to the irradiation chamber, which was
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connected to an ice bath. Irradiation conditions were those described above for growing cultures except that the temperature was maintained at 6–8 °C. The optical density of the cultures remained unchanged at a value close to 0.2 during irradiation at low temperature. Immediately after irradiation bacteria were harvested by centrifugation and stored at )20 °C until they were processed for tRNA extraction and analysis. 2.4. Measurements of s4 U and Pyo(4–5)Cyt contents in tRNA The procedures followed for tRNA extraction and hydrolysis, as well as the protocol for reverse phase HPLC analysis of the hydrolysis products has been described previously [13]. The s4 U and Pyo(4–5)Cyt contents were estimated by the areas under the peaks corresponding to these compounds in the chromatograms obtained by HPLC analysis of hydrolysed tRNA. To account for differences in the amount of tRNA present in the samples, the areas under the peaks corresponding to guanosine or guanine were also measured. The results were expressed as the ratio between the areas under the peaks corresponding to s4 U and guanosine, or Pyo(4–5)Cyt and guanine, in the same chromatogram. The values obtained in this way are proportional to the ratio between the concentration of tRNA carrying s4 U, or Pyo(4–5)Cyt, and the total tRNA concentration in each sample. 2.5. Estimation of rate constants for Pyo(4–5)Cyt formation The formation of Pyo(4–5)Cyt follows a pseudo first order kinetics both in vitro [14,15] and in vivo [16–21]. Since in bacteria maintained at 6–8 °C total tRNA concentration remained unchanged, the modifications in the s4 U and Pyo(4–5)Cyt contents during irradiation in this condition depended on photoproduct formation only. In order to calculate the rate constant for the cross-linking reaction at low temperature, a non-linear regression computer program was employed. Using this program we fit the parameters of the following equation to the experimental data obtained during the measurements of s4 U content: cs ¼ co expðktÞ þ cn ;
ð1Þ
where cs is the s4 U content after an irradiation time t, co and cn are the contents of s4 U available and non-available for cross-linking respectively at the start of the reaction, and k is the pseudo first order rate constant. The value of k was also calculated from Pyo(4–5)Cyt accumulation data, fitting the parameters of the following equation to the data obtained: cp ¼ cf ð1 expðktÞÞ;
ð2Þ
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where cp is the Pyo(4–5)Cyt content after an irradiation time t, and cf is the Pyo(4–5)Cyt content after complete reaction. When bacteria were irradiated during the exponential growth phase, measurements of s4 U and Pyo(4–5)Cyt contents were affected by tRNA synthesis and thiolation, in addition to photoproduct formation. As an approximation to analyze the results obtained under these conditions, we assumed that s4 U synthesis stopped quickly when irradiation started and was negligible during UVA exposure. The rate of tRNA accumulation during culture growth is expected to be a function of the rate of stable RNA accumulation [17]. In exponentially growing E. coli irradiated at 366 nm, the effects of radiation on the increase in optical density and on the RNA accumulation were closely similar [18]. In order to take into account the influence of changes in total tRNA concentration on the measurements of s4 U and Pyo(4– 5)Cyt content, a correction, based on the increase in optical density of the culture, was introduced in the calculations. Results obtained with bacteria irradiated at 37 °C were used to fit the parameters of the following equations to experimental data: cs ¼ ðco expðktÞ þ cn Þ=ðODt =ODo Þ;
ð3Þ
cp ¼ ðcf ð1 expðktÞÞÞ=ðODt =ODo Þ;
ð4Þ
where ODt and ODo are the optical densities after an irradiation time t and at the onset of irradiation respectively. The meaning of the other parameters is the same as were described for Eqs. (1) and (2). The optical density expected during irradiation was approximated by the polynomial ODt ¼ 0:149 þ 2:70 104 t 6:26 106 t2 þ 2:33 108 t3 :
ð5Þ
The coefficients included in Eq. (5) were obtained from a typical growth curve by non-linear fitting.
3. Results 3.1. Effect of UVA on tRNA sulfurtransferase activity In order to investigate the effect exerted by UVA on s4 U synthesis, tRNA sulfurtransferase activity was measured in E. coli extracts obtained from irradiated and non-irradiated control cells. When the products obtained after incubation of extracts with sulfur-deficient tRNA, labelled cysteine, and the required cofactors were analyzed, the results shown in Fig. 1 were obtained. Sulfur incorporation found in the extract prepared from irradiated cells was barely one fourth of that observed in the extract obtained from control cells. These results indicate a striking reduction in tRNA sulfurtranferase activity in E. coli after the UVA treat-
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Fig. 1. Sulfur transference from cysteine to tRNA. Sulfur deficient tRNA was incubated with 35 S labelled cysteine, ATP, Mg2þ , and extracts from non-irradiated (panel a) or irradiated (panel b) bacteria. After incubation tRNA was phenol extracted, ethanol precipitated, and deacylated. Finally, tRNA was purified by chromatography on Sephadex G-100. The collected fractions were monitored for absorbance at 260 nm (open symbols) and radioactivity (solid symbols).
ment. Taking into account that 68% of the sulfur contained in S. typhimurium tRNA is incorporated as s4 U [22], a remarkable reduction in the synthesis of this thionucleoside as a consequence of UVA exposure is inferred. 3.2. tRNA modification during growth under UVA irradiation To test whether the effect described in the preceding section could have some influence on the induction of growth delay under physiological conditions, we studied the changes in the tRNA contents of s4 U and Pyo(4– 5)Cyt induced by UVA in exponentially growing E. coli irradiated at a fluence rate similar to that expected in natural environments. The effect of irradiation on the culture growth is shown in Fig. 2. In keeping with data reported for cultures of E. coli exposed to UVA at low fluence rates [6,18], the growth rate decreased without complete cessation of growth, and when irradiation was prolonged beyond 100 min the cultures seemed to grow exponentially but with a longer cell mass doubling time. Radiation effects on tRNA modification are shown in Fig. 3. As a reference, changes in s4 U and Pyo(4–5)Cyt tRNA contents were studied in bacteria irradiated under similar conditions but maintained at low temperature to stop bacterial growth and avoid any effect related with tRNA turnover. As shown in Fig. 3a, in growing cells the reduction in the s4 U content during irradiation was faster than that observed in non-growing cells. The re-
Fig. 2. Effect of UVA irradiation on bacterial growth. Bacteria were incubated in minimal medium at 37 °C. The cultures were maintained in the dark (open circles) or irradiated at 365 nm (solid circles). In the culture exposed to UVA, irradiation started when the optical density reached a value of 0.15 (arrow). Eq. (5) is also plotted.
maining s4 U content in bacteria irradiated at 6–8 °C was always higher than that measured for bacteria incubated at 37 °C and exposed to UVA during the same time. In keeping with several reports on the kinetics of Pyo(4– 5)Cyt formation [14,16], accumulation of Pyo(4–5)Cyt in non-growing cells was concomitant with s4 U disappearance (Figs. 3a and b). After 60 min irradiation at low temperature the photoproduct content was similar to that found when a solution of E. coli tRNA 0.7 mg/ml in sodium cacodylate 0.05 M, pH 7, NaCl 0.1 M was irradiated at 365 nm at a total fluence of 155 kJ m2 . By fitting Eqs. (1) and (2) to experimental data obtained with bacteria maintained at 6–8 °C, the half-times for s4 U content decrease and Pyo(4–5)Cyt accumulation were estimated to be 10.1 and 11.5 min respectively, and 78% of the of s4 U was estimated to be available for cross-linking in this condition. The progress of photoproduct accumulation in growing bacteria seemed remarkably different from that observed at low temperature (Fig. 3b). The Pyo(4–5)Cyt content increased during the first 30 min of UVA exposure, reaching a maximum value corresponding to about 70% of the photoproduct accumulated in nongrowing bacteria after complete reaction. When irradiation was prolonged (for 60 min or more) a reduction in the tRNA Pyo(4–5)Cyt content was observed. By fitting Eqs. (3) and (4) to experimental data obtained at 37 °C, the half-times for s4 U content decrease and Pyo(4–5)Cyt accumulation in this condition were estimated to be 9.9 and 10.2 min respectively, and 71% of the s4 U present in tRNA seemed to be available for cross-linking.
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Fig. 3. Changes in tRNA contents of s4 U and Pyo(4–5)Cyt in bacteria grown under UVA irradiation. Cultures were maintained in the dark (open symbols) or exposed to radiation (solid symbols), while they were growing at 37 °C (circles) or after cooling at 6–8 °C (triangles). At the indicated times bacteria were harvested and tRNA was extracted. The contents of s4 U (panel a) and Pyo(4–5)Cyt (panel b) were measured by reverse phase HPLC analysis of hydrolysed tRNA. Eqs. (1) (panel a, dashed line), (2) (panel b, dashed line), (3) (panel a, solid line), and (4) (panel b, solid line) are also plotted. The values assigned to the parameters involved in these equations were estimated by fitting them to experimental data by non-linear regression. The arbitrary units used in panels a and b are different, by comparing the changes in tRNA contents of s4 U and Pyo(4–5)Cyt after complete reaction in nongrowing conditions it was estimated that 1 A.U. in panel a is equivalent to 0.87 A.U. in panel b.
Since the estimations of the half time for the reaction in both irradiation conditions are similar, and the contents of s4 U and Pyo(4–5)Cyt measured at 37 °C are comparable with the values calculated using Eqs. (3) and (4), the inactivation of s4 U synthesis during irradiation seems to be a suitable approximation to explain our results.
4. Discussion The tRNA-sulfurtransferase activity measurements in UVA-irradiated and non-irradiated bacteria showed that, as we suggested previously [8], the synthesis of s4 U is an UVA-sensitive process. The unique function attributed to s4 U is the triggering of the UVA-induced growth delay, and it is generally accepted that this modified residue acts as a sensor for UVA exposure in bacterial tRNA. In irradiated bacteria, the effectiveness
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of any process related to the growth delay could be conditioned by the ability of UVA to reduce the intracellular concentration of tRNA molecules carrying s4 U. The inhibition of tRNA thiolation is probably a relevant effect in physiological conditions, because it could be induced by low doses of radiation delivered at a low fluence rate. When bacteria are irradiated in non-growing conditions, the cross-linking process involves s4 U synthesized during pre-irradiation growth and the effect of UVA on s4 U synthesis is undetectable. A different situation may be expected when growing cells are exposed to UVA, since in this condition tRNA synthesis and photo-induced cross-linking take place simultaneously, during irradiation. Studying the influence of the dose rate on the induction of growth delay in exponentially growing E. coli cells, Favre [6] proposed a model to relate growth rate and concentration of intact target molecules. According to this model, the extent of the tRNA inactivation depends on the balance between UVA-induced crosslinking of tRNA and de novo synthesis of UVA-sensitive tRNA, so changes in growth rate are explained by the extent of Pyo(4–5)Cyt accumulation in tRNA [6,18]. In our assay conditions, no evident correlation between growth rate and Pyo(4–5)Cyt accumulation was found (Figs. 2 and 3b). Moreover, if during irradiation damaged tRNA were ‘‘diluted’’ with tRNA carrying s4 U, a decrease in Pyo(4–5)Cyt content should be accompanied by a simultaneous increase in s4 U content and, as shown in Fig. 3, this was not the case. The limited accumulation of Pyo(4–5)Cyt and the lack of correlation between variations of s4 U and Pyo(4–5)Cyt contents in growing bacterial cultures exposed to UVA, could be interpreted assuming that UVA inhibits s4 U synthesis in addition to inducing the cross-linking reaction. Experimental data suggest that in growing bacteria exposed to UVA Pyo(4–5)Cyt is formed mainly by reaction of s4 U synthesized before irradiation, and that tRNAs carrying either s4 U or Pyo(4–5)Cyt are ‘‘diluted’’ during irradiation with tRNA lacking s4 U. The tRNA molecules lacking s4 U are biologically active [23,24] but not UVA sensitive. The inhibition of s4 U synthesis should not interfere with protein synthesis, but it would reduce the concentration of the target molecules involved in the first event of the growth delay mechanism. The lack of UVA-sensitive tRNA would limit the accumulation of inactivated tRNA, conditioning the ability of UVA radiation to trigger the growth delay. The concordance between the experimental data and the s4 U and Pyo(4–5)Cyt contents calculated assuming that s4 U synthesis is inhibited during UVA exposure supports this hypothesis. Caldeira de Araujo and Favre [18] reported that in growing cultures of E. coli submitted to UVA at low fluence rate only 80% of the s4 U available for
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cross-linking was converted to Pyo(4–5)Cyt after a prolonged irradiation. Similarly, our calculations estimated that the fraction of s4 U available for cross-linking in growing bacteria was 71% instead of 78% as for nongrowing bacteria. The difference in the availability of s4 U for cross-linking cannot be explained by the inhibition of tRNA thiolation. After 120 min irradiation the content of Pyo(4–5)Cyt decreased faster than expected by dilution of cross-linked tRNA with tRNA synthesized de novo, and the reason for this fact is unknown. An abrupt decrease in the Pyo(4–5)Cyt tRNA content has been described in growing E. coli irradiated at 366 nm with a high fluence rate, and based on this observation the existence of a repair mechanism for crosslinked tRNA was proposed [7]. This repair mechanism has never been demonstrated, but its eventual existence could explain the deviation observed in our experimental data from the expected values at the end of irradiation. On the other hand, growth inhibition described in cultures irradiated at high fluence rates [6] could be produced by quick transformation of previously synthesized s4 U in Pyo(4–5)Cyt. Probably the inactivation of most UVA-sensitive tRNA in a short time could not be compensated by tRNA synthesis. Recent studies performed by Mueller and co-workers [25–27] and by Kambampati and Lauhon [28–30] demonstrated the involvement of the proteins ThiI and IscS in the sulfur transfer from cysteine to s4 U in E. coli. IscS contains pyridoxal phosphate as a cofactor and the absorbance spectrum of the purified protein in its reduced form exhibits a maximum at 363 nm [31]. This characteristic suggests that IscS could be the target for inactivation of s4 U synthesis during irradiation at 365 nm. Both ThiI and IscS are involved in thiamin biosynthesis [30,32] and strains carrying iscS mutations require nicotinic acid in addition to thiamin [30]. To test whether the effect of UVA on the growth rate was related with requirements associated with a deficient activity of IscS, an irradiation assay was performed on a culture growing in minimal medium supplemented with thiamin and nicotinic acid. The effect of UVA on bacteria growing in this supplemented medium was similar to that found in non-supplemented medium (data not shown), and no evidence has been found for the role of IscS in the effect of UVA on s4 U synthesis. It has been proposed that the growth delay protects bacteria against harmful effects of ultraviolet radiation and sunlight [33], but discrepant results were reported when the UVA sensitivity of mutants lacking s4 U was compared with that found in wild type strains. These mutants exhibit increased [34] or reduced [9,35] resistance to the lethal effects of UVA depending on the irradiation conditions. It has been suggested that s4 U is involved in the generation of DNA single strands breaks by UVA [35]. Under certain irradiation conditions, a transient lack of s4 U could help bacteria to avoid dele-
terious effects of UVA related to absorption of radiation by s4 U, increasing survival even when the induction of growth delay and the efficiency of any protective mechanism dependent on this effect is reduced.
Acknowledgements We thank Dr. D.N. Ant on for the bacterial strain ~ez for technical askindly provided, and Mr. J.A. Iban sistance. This research was supported in part by a grant from Fundaci on Balseiro.
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