- Email: [email protected]
Influence of rpoS mutations on the response of Salmonella enterica serovar Typhimurium to solar radiation
Journal of Photochemistry and Photobiology B: Biology 102 (2011) 20–25
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Influence of rpoS mutations on the response of Salmonella enterica serovar Typhimurium to solar radiation Oscar J. Oppezzo ⇑, Cristina S. Costa, Ramón A. Pizarro Comisión Nacional de Energía Atómica, Departamento de Radiobiología, Argentina
a r t i c l e
i n f o
Article history: Received 15 June 2010 Received in revised form 3 August 2010 Accepted 30 August 2010 Available online 7 September 2010 Keywords: Salmonella rpoS Sunlight Photodamage Survival
a b s t r a c t Salmonella enterica serovar Typhimurium is an important pathogen, and exhibits considerable resistance to the lethal effects of solar radiation. To evaluate the involvement of the RpoS transcription factor in the defense mechanisms of this organism, the sunlight response of a wild type strain (ATCC14028) was compared with that of an rpoS mutant, which exhibited increased sensitivity. Kinetics of cell death was complex in both strains, probably due to the presence of a variety of targets for the radiation. When ultraviolet radiation was excluded from the incident sunlight, lethal effects were abolished independently of the allelic state of rpoS. Reduction of oxygen concentration in the irradiation medium provided moderate protection to ATCC14028, but notably improved survival of the mutant. Similar assays were developed with another S. enterica strain (DA1468), which is a derivative of strain LT2 and produces low levels of RpoS. In this strain the loss of viability reveals the dependence on solar ultraviolet and oxygen concentration found for ATCC14028, but radiation resistance was slightly reduced. Increased sensitivity was observed in an rpoS mutant derived from DA1468, indicating that RpoS functions related to photoprotection are conserved in this strain. In addition, notable differences in the shape of the survival curves obtained for mutants derived from ATCC14028 and DA1468 were found, suggesting that genes beyond RpoS control are relevant in the sunlight response of these mutants. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction Solar radiation has long been recognized as a deleterious agent challenging survival of bacteria in natural environments [1,2]. A detailed knowledge of the effects of sunlight on bacteria is relevant to understand the fate of these organisms in natural waters [3], and could be helpful to design treatment techniques to improve microbiological quality of water by sunlight exposure [4]. Considerable differences in radiation sensitivity were reported among Gram negative bacterial species when their responses to natural sunlight [5,6] and artificial ultraviolet A radiation [6–8] were compared, and a remarkable radiation resistance was found in Salmonella enterica serovar Typhimurium [5,6], hereafter designated Salmonella Typhimurium. Considering that some strains of S. Typhimurium are important pathogens [9], further studies concerning the response of this bacterium to solar radiation would be of interest. The RpoS transcription factor controls the expression of a number of genes involved in responses to environmental stress in bacteria [10], including structural genes for DNA-binding proteins ⇑ Corresponding author. Address: Comisión Nacional de Energía Atómica, Departamento de Radiobiología, Avenida General Paz 1499, B1650KNA General San Martín, Buenos Aires, Argentina. Tel.: +54 11 6772 7013; fax: +54 11 6772 7188. E-mail address: [email protected] (O.J. Oppezzo). 1011-1344/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2010.08.012
believed to protect DNA from oxidative stress and enzymes involved in prevention of oxidative damage. The first phenotype described in rpoS mutants was an increased sensitivity to near ultraviolet radiation [11], and it has been reported that rpoS mutations increase sunlight sensitivity in Pseudomonas syringae [12], S. Typhimurium [13], and Escherichia coli [14]. A description of the effects of rpoS inactivation in S. Typhimurium could help to understand the unusual sunlight resistance observed in this bacterium. In the present study, the sunlight responses of wild type and rpoS mutant strains of S. Typhimurium were compared. As a first characterization of the mechanisms involved in the loss of bacterial viability, the contribution of solar ultraviolet to the induction of lethal effects was tested, and the dependence of the kinetics of cell death on oxygen concentration was evaluated. The ability of RpoS to provide photoprotection was also assayed in a S. Typhimurium strain carrying a rare TTG start codon in the rpoS gene. This mutation results in a low level of RpoS [15,16], contributing to avirulence [17,18], and increasing the sensitivity of the cells to DNA damage and acid stress, without appreciable effect on their tolerance to starvation and oxidative stress [17]. An influence of the genetic context on the survival of rpoS mutants is suggested, based on the comparison of the effects produced by rpoS inactivation in different strains.
O.J. Oppezzo et al. / Journal of Photochemistry and Photobiology B: Biology 102 (2011) 20–25
2. Materials and methods 2.1. Bacterial strains S. enterica serovar Typhimurium ATCC14028 [19] was used as the wild type. Strain PB4076 is a derivative of ATCC14028 carrying an rpoS deletion generated by one step inactivation [20], and was kindly provided by F.C. Soncini (Instituto de Biología Molecular y Celular de Rosario, Rosario, Argentina). Strain DA1468 is a derivative of the avirulent S. Typhimurium LT2 strain carrying an argC95 mutation [21]. Strain DA2245 was obtained by introducing the mutation rpoS::ApR in DA1468 by transduction with phage P22 HT105/1 int-201 grown on strain SF1005 [22], using resistance to ampicillin for positive selection. 2.2. Culture conditions Nutrient broth (8 g Difco nutrient broth, 5 g NaCl per liter) was sterilized by autoclaving and used for cultivation. Bacteria from stock cultures were loop inoculated in nutrient broth and incubated for 24 h at 37 °C with shaking. Aliquots of these cultures were diluted with fresh medium and incubated for an additional 24 h under the same conditions. Cells in stationary phase were harvested by centrifugation (8000g, 8 min, 20 °C) and used in sunlight response assays. 2.3. Sunlight irradiation Assays were performed on the roof of the laboratory (34°340 S 58°300 W), on cloudless days, at noon. Bacteria were washed and suspended in 0.15 M NaCl (approximately 108 colony forming units ml1), and exposed to sunlight in 1 cm path quartz spectrophotometer cells (1 1 4.5 cm). The quartz cells were placed in an aluminum holder which covered their top, bottom, and lateral sides but left their frontal and back faces free. When required, the frontal face of the cells was covered with a polycarbonate sheet (LexanÒ, General Electric) to exclude solar ultraviolet from the incident radiation, or with an opaque material to shield the samples from irradiation and use them as dark controls. The temperature of the samples was controlled by water circulation through the cell holder and monitored with an YSI-42SC tele-thermometer provided with a 9545-C15 probe (Arthur Thomas, Philadelphia, PA). For 15 min before and during the irradiations air was bubbled through the bacterial suspensions in order to ensure aeration and homogenization. When indicated, samples were bubbled with nitrogen instead of air to reduce the oxygen concentration in the irradiation medium. Solar irradiance was measured with an ILT1400-A radiometer equipped with a SEL033/QNDS2/W sensor (International Light Technologies, Peabody, MA) attached to the cell holder, and calibrated against a Rho Sigma photovoltaic radiometer. The fluence rates for sunlight irradiation measured with this instrument refer to the wavelength range of 200–1100 nm. The holder was equipped with a fork type equatorial mount in order to maintain the frontal faces of the quartz cells perpendicular to the direction of the solar disk during irradiations. 2.4. Enumeration procedure and data analysis At the onset of the experiments and at regular intervals during irradiations samples were removed and diluted in decimal steps with sterile 0.15 M NaCl solution. Aliquots of suitable dilutions were spread on nutrient agar plates and colonies were counted after incubation at 37 °C in the dark for 40 h. Since measurements of total solar irradiance were approximately constant during the exposures, with maximal changes of
21
5% during a complete experiment, the imparted dose was assumed to be proportional to the irradiation time for a given sample. Survival curves were analyzed using the target theory model [23]. Bacteria contain a variety of photosensitive molecules [24] and therefore, a variety of potential targets for the action of sunlight. It was assumed that in an irradiated bacterial population, some cells could lose viability due to the inactivation of one kind of target, with kinetics similar to that predicted by the single-target single-hit model. Simultaneously, damage could accumulate due to the inactivation of another kind of target, killing other cells with kinetics comparable to that predicted by the multi-target single-hit model when a threshold level of damage is reached. The survival probability per cell would be the product of the survival probabilities per cell corresponding to each inactivation process, whose logarithms are given by the terms on the right hand side of the equation:
logðN=N0 Þ ¼ logðeqt Þ þ logð1 ð1 ekt Þn Þ
ð1Þ
This expression relates the survival fraction (N/N0) to the imparted dose (t). The first term accounts for cell killing occurring with single-hit single-target kinetics, and q is the inverse of the dose required for 1/e (37%) survival from this killing process. The second term accounts for a loss of viability occurring with single-hit multiple-target kinetics, and the parameter k is the inverse of the dose required for 1/e survival from accumulative damage when the imparted dose exceeds a threshold value. A single cell is expected to survive this damage until the inactivation of a number of targets proportional to parameter n, and the quasi-threshold dose (tq) is related with k and n by: 1
tq ¼ k
lnðnÞ
ð2Þ
Alternatively, a model proposed for biphasic microbial survival curves [25] was used. The equation corresponding to this model is:
logðN=N0 Þ ¼ logðfe
q1t
þ ð1 f Þeq2t Þ
ð3Þ
where f is the fraction of the initial population in a major subpopulation, (1 f) is the fraction of the initial population in a minor subpopulation which is more resistant than the previous one, and q1 and q2 are the inverse of the doses required for 1/e survival of the two populations, respectively. In this case the single-target singlehit model is used to describe the inactivation of each subpopulation. The parameters of the corresponding equations were fitted to the experimental data using a non-lineal regression program (Microcal Origin 6.0). 3. Results 3.1. Sunlight response of the wild type strain Under the irradiation conditions used, the sensitivity of the strain ATCC14028 to natural solar radiation was low (Figs. 1–3). Survival curves obtained with unshielded samples seem linear, except for an increase in the slope at the end of the irradiations. The time required for 1/e survival in the initial portion of the curves was 106 (±29) min. A comparable value of 82 min can be calculated from reported data for ATCC14028 exposed to sunlight under similar conditions [5]. The parameters of Eq. (1) can be fitted to experimental data obtained during irradiations of ATCC14028, suggesting that simultaneous inactivation of different targets could be responsible for the shape of the survival curves. When samples were shielded with a material which excludes 12% of the visible light and most of radiation with wavelengths below 390 nm from the incident sunlight (Fig. 1 inset), viable counts remained unchanged during the irradiation, resembling the results obtained for dark controls (Fig. 1). Solar ultraviolet seems therefore
22
O.J. Oppezzo et al. / Journal of Photochemistry and Photobiology B: Biology 102 (2011) 20–25
0
0
-2
log (N/N0)
100
-1
log (N/N0)
T%
80 60 40
-4
20 0
-6 400
800
1200
(nm) 0
60
120
0
180
60
120
Irradiation time (min)
Irradiation time (min) Fig. 1. Influence of solar ultraviolet on survival of strain ATCC14028 exposed to natural sunlight. A bacterial suspension was bubbled with air and irradiated in quartz cells unshielded (open circles), covered with a polycarbonate sheet (open squares), or covered with an opaque material (close circles). The function obtained by fitting the parameters of Eq. (1) to the experimental results for the unshielded sample (solid line) is also plotted. Irradiation start time was 2010-03-22-15:30 (Coordinated Universal Time, UTC) (11.30 a.m.), irradiance was 847–861 W m2, and temperature of the samples was 26 °C. Inset: absorption spectrum of the polycarbonate sheet used to cover selected samples. The experiment was repeated twice, representative results are shown.
Fig. 3. Effect of rpoS mutations on survival of Salmonella enterica serovar Typhimurium exposed to natural sunlight. Suspensions of strains ATCC14028 (open circles), DA1468 (open pentagons), and PB4076 (open diamonds) were irradiated in unshielded quartz cells while bubbled with air. The functions obtained by fitting the parameters of Eq. (1) to the experimental results for ATCC14028 (solid line), DA1468 (dashed line), and PB4076 (dotted line) were also plotted. Irradiation start time was 2010-02-08-15:40 (UTC), irradiance was 922–928 W m2, and temperature of the samples was 29 °C. The experiment was repeated three times, representative results are shown.
Reduction of the oxygen concentration in the irradiation medium had limited influence on survival of ATCC14028 at the start of the exposure, but this condition seemed to protect the bacteria when the irradiation was prolonged (Fig. 2). This result strongly suggests that the increase in the rate of cell death observed when cells were bubbled with air depends on the photodynamic action of radiation.
log (N/N0)
0
3.2. Effects of rpoS mutations Comparison of the survival curves obtained for the strains ATCC14028 and PB4076 demonstrated that the deletion of the rpoS gene produces a considerable increase in the sunlight sensitivity of S. Typhimurium (Fig. 3). The dose required to reduce viable counts of the mutant to the detection limit allowed for survival of one third of the initial bacterial population of the wild type strain. The parameters of Eq. (1) can be fitted to the experimental data
-1
0
60
120
Irradiation time (min) Fig. 2. Influence of oxygen concentration on survival of strain ATCC14028 exposed to natural sunlight. A bacterial suspension was irradiated in unshielded quartz cells while bubbled with air (open circles) or nitrogen (open triangles). The function obtained by fitting the parameters of Eq. (1) to the experimental results for the sample bubbled with air (solid line), and that obtained by fitting the parameters of the first term in Eq. (1) to the experimental results for the sample bubbled with nitrogen (dashed line) were also plotted. Irradiation start time was 2010-02-2515:25 (UTC), irradiance was 881–894 W m2, and temperature of the samples was 26 °C. The experiment was repeated twice, representative results are shown.
to play a key role to induce the lethal effects observed in ATCC14028.
Table 1 Comparison of the sunlight susceptibility of different S. Typhimurium strains. Strain ATCC14028 PB4076 DA1468
1/q (J m2)a 6
5.75 10 (1.01 106) 1.22 106 (2.62 105) 4.19 106 (9.81 105)
1/k (J m2)a 5
8.43 10 (3.32 105) 2.49 105 (6.95 104) 4.75 105 (1.27 105)
tq (J m2)a 6.60 106 (7.50 105) 3.08 106 (1.30 106) 6.12 106 (2.71 105)
a Parameters of Eq. (1) were fitted to the experimental survival curves. Using the obtained values and solar irradiation data for the wavelength range between 200 and 1100 nm, the fluences required for 1/e (37%) survival from the single-hit (1/q) and multiple-hit (1/k) killing processes, and the fluence equivalent to the quasithreshold dose (tq), were calculated. Mean values from three independent measurements are shown, and the corresponding standard deviations are given in parenthesis.
23
0
0
-2
-2
log (N/N0)
log (N/N0)
O.J. Oppezzo et al. / Journal of Photochemistry and Photobiology B: Biology 102 (2011) 20–25
-4
-4
-6 -6 0
60
120
0
Irradiation time (min) Fig. 4. Influence of solar ultraviolet and oxygen concentration on survival of strain PB4076 exposed to natural sunlight. A bacterial suspension was irradiated in unshielded quartz cells while bubbled with air (open circles) or nitrogen (open triangles), and in quartz cells covered with a polycarbonate sheet (open squares) or with an opaque material (close circles) while bubbled with air. The functions obtained by fitting the parameters of Eq. (1) to the experimental results for the unshielded samples bubbled with air (solid line) and nitrogen (dashed line) were also plotted. Irradiation start time was 2010-03-26-15:30 (UTC), irradiance was 854–874 W m2, and temperature of the samples was 26 °C. The experiment was repeated twice, representative results are shown.
obtained with PB4076. In this strain the initial slope (q) and the additional slope in the final portion of the curve (k) were found increased, and the dose corresponding to the change in the slope (calculated as the quasi-threshold dose) was reduced with respect to the values estimated for the strain ATCC14028 (Table 1). It should be noted that the survival curves for PB4076 could also be modeled using a Weibull type model proposed for convex inactivation curves [25], which can be reduced to the expression of a simple exponential decay elevated to an exponent whose physical meaning is undefined. Since this model is not suitable to describe inactivation of ATCC14028, the rpoS mutation might slightly modify the shape of the survival curve, and a comparison of the numerical values estimated for the parameters of Eq. (1) would not allow a complete description of the effects of this mutation. Similarly to the wild type strain, lethal effects were undetectable in PB4076 when the ultraviolet component was excluded from the incident radiation (Fig. 4). Since both, wild type and mutant bacteria were resistant to the effects of visible light, it may be inferred that RpoS is required to overcome the effects of ultraviolet radiation during sunlight exposures. Nitrogen bubbling provided considerable protection to the mutant and this effect could be observed even at low doses (Fig. 4), suggesting that the increment in lethal effect in the rpoS mutant depends to a great extent on the photodynamic action of the radiation. The same experimental approach was used with a S. Typhimurium strain derived from LT2, carrying the rpoS mutation present in this strain. Survival curves obtained for the strain DA1468 had a shape similar to that of the curve for ATCC14028 (Fig. 3). Fitting the parameters of Eq. (1) to the experimental data the value for k was found increased in DA1468 with respect to that estimated for ATCC14028 (Table 1). When suspensions of DA1468 were shielded against solar ultraviolet or bubbled with nitrogen, the changes observed in the survival curves were identical to those found with ATCC14028 (data not shown). Conclusions about the
60
120
Irradiation time (min) Fig. 5. Effect of rpoS inactivation on survival of strain DA1468 exposed to natural sunlight. Suspensions of strains DA1468 (open pentagons), and DA2245 (open inverted triangles), were irradiated in unshielded quartz cells while bubbled with air. The function obtained by fitting the parameters of Eq. (1) to the experimental results for DA1468 (solid line) and that obtained by fitting the parameters of Eq. (3) to the experimental results for DA2245 (doted line) were also plotted. Irradiation start time was 2010-03-16-15:54 (UTC), irradiance was 860–866 W m2, and temperature of the samples was 25 °C. The experiment was repeated twice, representative results are shown.
importance of solar ultraviolet and the contribution of the photodynamic action to the lethal effects of sunlight on ATCC14028 can be extended to DA1468, suggesting that the increased sensitivity found in these strains is related with the damage induced by reactive oxygen species. To estimate the contribution of systems under RpoS control to the survival of DA1468 exposed to sunlight, the response of this strain was compared with that of its derivative DA2245, which carries an insertion inactivating rpoS. Increased sensitivity found in DA2245 compared to DA1468 demonstrates that RpoS functions related with photoprotection are conserved in strains derived from LT2 (Fig. 5). The shape of the survival curve for DA2245 was very different to that found for PB4076. No shoulder was apparent and the rate of cell death decreased when the irradiation was prolonged producing a tail and suggesting the presence of a bacterial subpopulation with increased resistance. 4. Discussion The strain ATCC14028 exhibited considerable sunlight tolerance, as described by other laboratories [5,6]. However, in contrast with log-linear survival curves previously reported for this strain [5], a complex cell death kinetics was observed. The sunlight response of ATCC14028 was dependent on oxygen concentration in the irradiation medium, and log-linear survival curves were obtained only at low oxygen concentrations. Differences in oxygen availability during the exposures could likely be the reason for the discrepancy between the results presented here and those reported by other researchers. A previous study concerning the influence of an rpoS mutation on the sunlight response of ATCC14028 was focused on bacterial survival in a marine environment [14], and the effects of radiation were assayed under experimental conditions similar to those expected in such an environment. Survival curves were obtained
24
O.J. Oppezzo et al. / Journal of Photochemistry and Photobiology B: Biology 102 (2011) 20–25
excluding solar ultraviolet from the incident radiation, and bacteria were suspended in seawater during irradiations. It is difficult to compare the results obtained under these conditions with those described here, since it has been shown that exposure to seawater increases the sensitivity of Salmonella wild type cells to natural sunlight [26], and that rpoS inactivation reduces the ability of S. enterica to tolerate the stress produced by seawater in the dark [27]. Interestingly, solar ultraviolet is required for the induction of lethal effects in ATCC14028, and RpoS seems to be involved in the defense systems against these effects, resembling the situation described in P. syringae, an organism adapted to a very different environment [12]. When ATCC14028 was irradiated under a nitrogen atmosphere the survival curve seemed linear in a semi-logarithmic plot, and exhibited a slope comparable to the initial slope in curve for cells bubbled with air (Fig. 2). This observation seems compatible with the model formulated in Eq. (1) assuming that the inactivation of targets responsible for cell killing with single-hit single-target kinetics is almost independent of the oxygen concentration in the irradiation medium, but the events responsible for accumulative lethal damage depend on this condition. Recent studies remark the role of oxidative damage to proteins in cell death induced by ultraviolet A radiation in E. coli [28,29]. A lost of viability produced by protein oxidation could exhibit single-hit multiple-target kinetics. If a single-hit were sufficient to inactivate a molecule of the target protein the conditions assumed in this model [23] would be fulfilled, because bacterial cells would contain many molecules of this protein, which would have the same probability of being inactivated, and inactivation of a single protein molecule should not produce cell death. The inactivation of a target protein, eventually a component of the respiratory chain [28], by reactive oxygen species generated during sunlight exposure could explain the cell death with single-hit multiple-target kinetics, the protective effect of nitrogen bubbling and, to some extent, the role of RpoS since in stationary phase this sigma factor controls the expression of genes involved in prevention of oxidative damage [10]. Nevertheless, the effect of the rpoS deletion on the survival curves of S. Typhimurium was not limited to the increase in the slope ascribed to the oxidative damage (Table 1 and Fig. 3), suggesting a more complex role for RpoS in the sunlight response of this bacterium. Whilst the survival curves obtained for PB4076 (Figs. 3 and 4) were comparable to those reported for E. coli rpoS mutants exposed to sunlight [5,14], the response of DA2245 was different (Fig. 5). Since RpoS is expected to be absent in both PB4076 and DA2245, differences in the survival curves of these strains could be attributed to their genetic backgrounds. In E. coli the expression of some genes involved in oxidative stress tolerance is controlled by both RpoS and OxyR, and the lack of one regulatory system can be partially compensated by the activity of the other [30]. If some genes related with sunlight response were expressed independently of RpoS in S. Typhimurium, the presence of different alleles of these genes in PB4076 and DA2245 could account for the differences in the radiation responses of these mutants. In conclusion, lethal effects induced by sunlight in S. Typhimurium depend on solar ultraviolet, and the contribution of the photodynamic action of the radiation to cell death is limited in the wild type strain, but relevant in rpoS mutants. Models used to analyze the action of solar radiation on S. Typhimurium should take into account the complex nature of this action to provide suitable descriptions of the survival curves. Since the genetic background of the rpoS mutant strains seems to modify the consequences of rpoS inactivation, descriptions of the influence of other genes on the sunlight response of S. Typhimurium would be necessary to understand the survival strategies of this organism.
Acknowledgments We thank F.C. Soncini for bacterial strains kindly provided. This work has been supported in part by a grant from Agencia Nacional de Promoción Científica y Tecnológica (PICT 38241). References [1] A.L.H. Gameson, J.R. Saxon, Field studies on effect of daylight on mortality of coliform bacteria, Water Res. 1 (1967) 279–295. [2] C.E. Chamberlin, R. Mitchell, in: R. Mitchell (Ed.), Water Pollution Microbiology, vol. 2, John Wiley and Sons, New York, 1978, pp. 325–348. [3] Y. Rozen, S. Belkin, Survival of enteric bacteria in seawater, FEMS Microbiol. Rev. 25 (2001) 513–519. [4] M. Wegelin, S. Canonica, K. Mechsner, T. Fleischmann, F. Pesaro, A. Metzler, Solar water disinfection: scope of the process and analysis of radiation experiments, J. Water Supply Res. Technol.-Aqua. 43 (1994) 154–169. [5] M. Berney, H.-U. Weilenmann, A. Simonetti, T. Egli, Efficacy of solar disinfection of Escherichia coli, Shigella flexneri, Salmonella Typhimurium and Vibrio cholerae, J. Appl. Microbiol. 101 (2006) 828–836. [6] F. Bosshard, M. Berney, M. Scheifele, H.-U. Weilenmann, T. Egli, Solar disinfection (SODIS) and subsequent dark storage of Salmonella Typhimurium and Shigella flexneri monitored by flow cytometry, Microbiology 155 (2009) 1310–1317. [7] R.O. Fernández, R.A. Pizarro, Lethal effect induced in Pseudomonas aeruginosa exposed to ultraviolet-A radiation, Photochem. Photobiol. 64 (1996) 334–339. [8] O.J. Oppezzo, R.A. Pizarro, Sublethal effects of ultraviolet A radiation on Enterobacter cloacae, J. Photochem. Photobiol. B: Biol. 62 (2001) 158–165. [9] M. Kennedy, R. Villar, D.J. Vugia, T. Rabatsky-Ehr, M.M. Farley, M. Pass, K. Smith, P. Smith, P.R. Cieslak, B. Imhoff, P.M. Griffin, Hospitalization and deaths due to Salmonella infections, FoodNet 1996–1999, Clin. Infect. Dis. 38 (3) (2004) S142–S148. [10] H. Weber, T. Polen, J. Heuveling, V.F. Wendisch, R. Hengee, Genome-wide analysis of the general stress response network in Escherichia coli: rsdependent genes, promoters, and sigma factor selectivity, J. Bacteriol. 187 (2005) 1591–1603. [11] R.W. Tuveson, R.B. Jonas, Genetic control of near-UV (300–400 nm) sensitivity independent of the recA gene in strains of Escherichia coli K12, Photochem. Photobiol. 30 (1979) 667–676. [12] C.D. Miller, W.S. Mortensen, G.U.L. Braga, A.J. Anderson, The rpoS gene in Pseudomonas syringae is important in surviving exposure to the near-UV in sunlight, Curr. Microbiol. 43 (2001) 374–377. [13] S. Zaafrane, K. Maatouk, J.M. Gauthier, A. Bakhrouf, Influence des conditions de culture préalables et de la présence du gène rpoS pour la survie de Salmonella Typhimurium en eau de mer exposée à la lumière solaire, Can. J. Microbiol. 50 (2004) 341–350. [14] M. Berney, H.-U. Weilenmann, J. Ihssen, C. Bassin, T. Egli, Specific growth rate determines the sensitivity of Escherichia coli to thermal, UVA, and solar disinfection, Appl. Environ. Microbiol. 72 (2006) 2586–2593. [15] L. Brown, T. Elliot, Efficient transcription of the RpoS sigma factor in Salmonella Typhimurium requires host factor I, an RNA-binding protein encoded by the hfq gene, J. Bacteriol. 178 (1996) 3763–3770. [16] V. Robbe-Saule, M. Dias Lopes, A. Kolb, F. Norel, Physiological effects of Crl in Salmonella are modulated by rs level and promoter specificity, J. Bacteriol. 189 (2007) 2976–2987. [17] M.R. Wilmes-Riesenberg, J.W. Foster, R. Curtiss, An altered rpoS allele contributes to the avirulence of Salmonella Typhimurium LT2, Infect. Immun. 65 (1997) 203–210. [18] W.E. Swords, B.M. Cannon, W.H. Benjamin, Avirulence of LT2 strains of Salmonella Typhimurium results from a defective rpoS gene, Infect. Immun. 65 (1997) 2451–2453. [19] T. Jarvik, C. Smillie, E.A. Groisman, H. Ochman, Short-term signatures of evolutionary change in the Salmonella enterica serovar Typhimurium 14028 genome, J. Bacteriol. 192 (2010) 560–567. [20] M.L. Cabeza, A. Aguirre, F.C. Soncini, E. García Véscovi, Induction of RpoS degradation by the two-component system regulator RstA in Salmonella enterica, J. Bacteriol. 189 (2007) 7335–7342. [21] O.J. Oppezzo, D.N. Antón, Involvement of cysB and cysE genes in the sensitivity of Salmonella Typhimurium to mecillinam, J. Bacteriol. 177 (1995) 4524–4527. [22] F.C. Fang, S.J. Libby, N.A. Buchmeier, P.C. Loewen, J. Switala, J. Harwood, D.G. Guiney, The alternative r factor KatF (RpoS) regulates Salmonella virulence, Proc. Natl. Acad. Sci. USA 89 (1992) 11978–11982. [23] E.L. Alpen, Radiation Biophysics, Academic Press, San Diego, 1998. [24] A. Eisenstark, in: J.G. Scandalios (Ed.), Advances in Genetics, vol. 26, Academic Press, San Diego, 1989, pp. 99–147. [25] A.H. Geeraerd, V.P. Valdramidis, J.F. Van Impe, GIanFiT, a free tool to assess non-log-linear microbial survivor curves, Int. J. Food Microbiol. 102 (2005) 95– 105. [26] C.M. Davies, L.M. Evison, Sunlight and survival of enteric bacteria in natural waters, J. Appl. Bacteriol. 70 (1991) 265–274. [27] P.M. Munro, G.N. Flatau, R.L. Clément, J.M. Gauthier, Influence of the RpoS (KatF) sigma factor on maintenance of viability and culturability of Escherichia coli and Salmonella Typhimurium in seawater, Appl. Environ. Microbiol. 61 (1995) 1853–1858.
O.J. Oppezzo et al. / Journal of Photochemistry and Photobiology B: Biology 102 (2011) 20–25 [28] F. Bosshard, M. Bucheli, Y. Meur, T. Egli, The respiratory chain is the cell’s Achilles’ heel during UVA inactivation in Escherichia coli, Microbiology 156 (2010) 2006–2015. [29] F. Bosshard, K. Riedel, T. Schneider, C. Geiser, M. Bucheli, T. Egli, Protein oxidation and aggregation in UVA irradiated Escherichia coli cells as signs of
25
accelerated cellular senescence, Environ. Microbiol., in press. doi:10.1111/ j.1462-2920.2010.02268.x. [30] A.B. Ivanova, G.V. Glinsky, A. Eisenstark, Role of rpoS regulon in resistance to oxidative stress and near-UV radiation in DoxyR suppressor mutants of Escherichia coli, Free Radic. Biol. Med. 23 (1997) 627–636.
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Influence of rpoS mutations on the response of Salmonella enterica serovar Typhimurium to solar radiation Oscar J. Oppezzo ⇑, Cristina S. Costa, Ramón A. Pizarro Comisión Nacional de Energía Atómica, Departamento de Radiobiología, Argentina
a r t i c l e
i n f o
Article history: Received 15 June 2010 Received in revised form 3 August 2010 Accepted 30 August 2010 Available online 7 September 2010 Keywords: Salmonella rpoS Sunlight Photodamage Survival
a b s t r a c t Salmonella enterica serovar Typhimurium is an important pathogen, and exhibits considerable resistance to the lethal effects of solar radiation. To evaluate the involvement of the RpoS transcription factor in the defense mechanisms of this organism, the sunlight response of a wild type strain (ATCC14028) was compared with that of an rpoS mutant, which exhibited increased sensitivity. Kinetics of cell death was complex in both strains, probably due to the presence of a variety of targets for the radiation. When ultraviolet radiation was excluded from the incident sunlight, lethal effects were abolished independently of the allelic state of rpoS. Reduction of oxygen concentration in the irradiation medium provided moderate protection to ATCC14028, but notably improved survival of the mutant. Similar assays were developed with another S. enterica strain (DA1468), which is a derivative of strain LT2 and produces low levels of RpoS. In this strain the loss of viability reveals the dependence on solar ultraviolet and oxygen concentration found for ATCC14028, but radiation resistance was slightly reduced. Increased sensitivity was observed in an rpoS mutant derived from DA1468, indicating that RpoS functions related to photoprotection are conserved in this strain. In addition, notable differences in the shape of the survival curves obtained for mutants derived from ATCC14028 and DA1468 were found, suggesting that genes beyond RpoS control are relevant in the sunlight response of these mutants. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction Solar radiation has long been recognized as a deleterious agent challenging survival of bacteria in natural environments [1,2]. A detailed knowledge of the effects of sunlight on bacteria is relevant to understand the fate of these organisms in natural waters [3], and could be helpful to design treatment techniques to improve microbiological quality of water by sunlight exposure [4]. Considerable differences in radiation sensitivity were reported among Gram negative bacterial species when their responses to natural sunlight [5,6] and artificial ultraviolet A radiation [6–8] were compared, and a remarkable radiation resistance was found in Salmonella enterica serovar Typhimurium [5,6], hereafter designated Salmonella Typhimurium. Considering that some strains of S. Typhimurium are important pathogens [9], further studies concerning the response of this bacterium to solar radiation would be of interest. The RpoS transcription factor controls the expression of a number of genes involved in responses to environmental stress in bacteria [10], including structural genes for DNA-binding proteins ⇑ Corresponding author. Address: Comisión Nacional de Energía Atómica, Departamento de Radiobiología, Avenida General Paz 1499, B1650KNA General San Martín, Buenos Aires, Argentina. Tel.: +54 11 6772 7013; fax: +54 11 6772 7188. E-mail address: [email protected] (O.J. Oppezzo). 1011-1344/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2010.08.012
believed to protect DNA from oxidative stress and enzymes involved in prevention of oxidative damage. The first phenotype described in rpoS mutants was an increased sensitivity to near ultraviolet radiation [11], and it has been reported that rpoS mutations increase sunlight sensitivity in Pseudomonas syringae [12], S. Typhimurium [13], and Escherichia coli [14]. A description of the effects of rpoS inactivation in S. Typhimurium could help to understand the unusual sunlight resistance observed in this bacterium. In the present study, the sunlight responses of wild type and rpoS mutant strains of S. Typhimurium were compared. As a first characterization of the mechanisms involved in the loss of bacterial viability, the contribution of solar ultraviolet to the induction of lethal effects was tested, and the dependence of the kinetics of cell death on oxygen concentration was evaluated. The ability of RpoS to provide photoprotection was also assayed in a S. Typhimurium strain carrying a rare TTG start codon in the rpoS gene. This mutation results in a low level of RpoS [15,16], contributing to avirulence [17,18], and increasing the sensitivity of the cells to DNA damage and acid stress, without appreciable effect on their tolerance to starvation and oxidative stress [17]. An influence of the genetic context on the survival of rpoS mutants is suggested, based on the comparison of the effects produced by rpoS inactivation in different strains.
O.J. Oppezzo et al. / Journal of Photochemistry and Photobiology B: Biology 102 (2011) 20–25
2. Materials and methods 2.1. Bacterial strains S. enterica serovar Typhimurium ATCC14028 [19] was used as the wild type. Strain PB4076 is a derivative of ATCC14028 carrying an rpoS deletion generated by one step inactivation [20], and was kindly provided by F.C. Soncini (Instituto de Biología Molecular y Celular de Rosario, Rosario, Argentina). Strain DA1468 is a derivative of the avirulent S. Typhimurium LT2 strain carrying an argC95 mutation [21]. Strain DA2245 was obtained by introducing the mutation rpoS::ApR in DA1468 by transduction with phage P22 HT105/1 int-201 grown on strain SF1005 [22], using resistance to ampicillin for positive selection. 2.2. Culture conditions Nutrient broth (8 g Difco nutrient broth, 5 g NaCl per liter) was sterilized by autoclaving and used for cultivation. Bacteria from stock cultures were loop inoculated in nutrient broth and incubated for 24 h at 37 °C with shaking. Aliquots of these cultures were diluted with fresh medium and incubated for an additional 24 h under the same conditions. Cells in stationary phase were harvested by centrifugation (8000g, 8 min, 20 °C) and used in sunlight response assays. 2.3. Sunlight irradiation Assays were performed on the roof of the laboratory (34°340 S 58°300 W), on cloudless days, at noon. Bacteria were washed and suspended in 0.15 M NaCl (approximately 108 colony forming units ml1), and exposed to sunlight in 1 cm path quartz spectrophotometer cells (1 1 4.5 cm). The quartz cells were placed in an aluminum holder which covered their top, bottom, and lateral sides but left their frontal and back faces free. When required, the frontal face of the cells was covered with a polycarbonate sheet (LexanÒ, General Electric) to exclude solar ultraviolet from the incident radiation, or with an opaque material to shield the samples from irradiation and use them as dark controls. The temperature of the samples was controlled by water circulation through the cell holder and monitored with an YSI-42SC tele-thermometer provided with a 9545-C15 probe (Arthur Thomas, Philadelphia, PA). For 15 min before and during the irradiations air was bubbled through the bacterial suspensions in order to ensure aeration and homogenization. When indicated, samples were bubbled with nitrogen instead of air to reduce the oxygen concentration in the irradiation medium. Solar irradiance was measured with an ILT1400-A radiometer equipped with a SEL033/QNDS2/W sensor (International Light Technologies, Peabody, MA) attached to the cell holder, and calibrated against a Rho Sigma photovoltaic radiometer. The fluence rates for sunlight irradiation measured with this instrument refer to the wavelength range of 200–1100 nm. The holder was equipped with a fork type equatorial mount in order to maintain the frontal faces of the quartz cells perpendicular to the direction of the solar disk during irradiations. 2.4. Enumeration procedure and data analysis At the onset of the experiments and at regular intervals during irradiations samples were removed and diluted in decimal steps with sterile 0.15 M NaCl solution. Aliquots of suitable dilutions were spread on nutrient agar plates and colonies were counted after incubation at 37 °C in the dark for 40 h. Since measurements of total solar irradiance were approximately constant during the exposures, with maximal changes of
21
5% during a complete experiment, the imparted dose was assumed to be proportional to the irradiation time for a given sample. Survival curves were analyzed using the target theory model [23]. Bacteria contain a variety of photosensitive molecules [24] and therefore, a variety of potential targets for the action of sunlight. It was assumed that in an irradiated bacterial population, some cells could lose viability due to the inactivation of one kind of target, with kinetics similar to that predicted by the single-target single-hit model. Simultaneously, damage could accumulate due to the inactivation of another kind of target, killing other cells with kinetics comparable to that predicted by the multi-target single-hit model when a threshold level of damage is reached. The survival probability per cell would be the product of the survival probabilities per cell corresponding to each inactivation process, whose logarithms are given by the terms on the right hand side of the equation:
logðN=N0 Þ ¼ logðeqt Þ þ logð1 ð1 ekt Þn Þ
ð1Þ
This expression relates the survival fraction (N/N0) to the imparted dose (t). The first term accounts for cell killing occurring with single-hit single-target kinetics, and q is the inverse of the dose required for 1/e (37%) survival from this killing process. The second term accounts for a loss of viability occurring with single-hit multiple-target kinetics, and the parameter k is the inverse of the dose required for 1/e survival from accumulative damage when the imparted dose exceeds a threshold value. A single cell is expected to survive this damage until the inactivation of a number of targets proportional to parameter n, and the quasi-threshold dose (tq) is related with k and n by: 1
tq ¼ k
lnðnÞ
ð2Þ
Alternatively, a model proposed for biphasic microbial survival curves [25] was used. The equation corresponding to this model is:
logðN=N0 Þ ¼ logðfe
q1t
þ ð1 f Þeq2t Þ
ð3Þ
where f is the fraction of the initial population in a major subpopulation, (1 f) is the fraction of the initial population in a minor subpopulation which is more resistant than the previous one, and q1 and q2 are the inverse of the doses required for 1/e survival of the two populations, respectively. In this case the single-target singlehit model is used to describe the inactivation of each subpopulation. The parameters of the corresponding equations were fitted to the experimental data using a non-lineal regression program (Microcal Origin 6.0). 3. Results 3.1. Sunlight response of the wild type strain Under the irradiation conditions used, the sensitivity of the strain ATCC14028 to natural solar radiation was low (Figs. 1–3). Survival curves obtained with unshielded samples seem linear, except for an increase in the slope at the end of the irradiations. The time required for 1/e survival in the initial portion of the curves was 106 (±29) min. A comparable value of 82 min can be calculated from reported data for ATCC14028 exposed to sunlight under similar conditions [5]. The parameters of Eq. (1) can be fitted to experimental data obtained during irradiations of ATCC14028, suggesting that simultaneous inactivation of different targets could be responsible for the shape of the survival curves. When samples were shielded with a material which excludes 12% of the visible light and most of radiation with wavelengths below 390 nm from the incident sunlight (Fig. 1 inset), viable counts remained unchanged during the irradiation, resembling the results obtained for dark controls (Fig. 1). Solar ultraviolet seems therefore
22
O.J. Oppezzo et al. / Journal of Photochemistry and Photobiology B: Biology 102 (2011) 20–25
0
0
-2
log (N/N0)
100
-1
log (N/N0)
T%
80 60 40
-4
20 0
-6 400
800
1200
(nm) 0
60
120
0
180
60
120
Irradiation time (min)
Irradiation time (min) Fig. 1. Influence of solar ultraviolet on survival of strain ATCC14028 exposed to natural sunlight. A bacterial suspension was bubbled with air and irradiated in quartz cells unshielded (open circles), covered with a polycarbonate sheet (open squares), or covered with an opaque material (close circles). The function obtained by fitting the parameters of Eq. (1) to the experimental results for the unshielded sample (solid line) is also plotted. Irradiation start time was 2010-03-22-15:30 (Coordinated Universal Time, UTC) (11.30 a.m.), irradiance was 847–861 W m2, and temperature of the samples was 26 °C. Inset: absorption spectrum of the polycarbonate sheet used to cover selected samples. The experiment was repeated twice, representative results are shown.
Fig. 3. Effect of rpoS mutations on survival of Salmonella enterica serovar Typhimurium exposed to natural sunlight. Suspensions of strains ATCC14028 (open circles), DA1468 (open pentagons), and PB4076 (open diamonds) were irradiated in unshielded quartz cells while bubbled with air. The functions obtained by fitting the parameters of Eq. (1) to the experimental results for ATCC14028 (solid line), DA1468 (dashed line), and PB4076 (dotted line) were also plotted. Irradiation start time was 2010-02-08-15:40 (UTC), irradiance was 922–928 W m2, and temperature of the samples was 29 °C. The experiment was repeated three times, representative results are shown.
Reduction of the oxygen concentration in the irradiation medium had limited influence on survival of ATCC14028 at the start of the exposure, but this condition seemed to protect the bacteria when the irradiation was prolonged (Fig. 2). This result strongly suggests that the increase in the rate of cell death observed when cells were bubbled with air depends on the photodynamic action of radiation.
log (N/N0)
0
3.2. Effects of rpoS mutations Comparison of the survival curves obtained for the strains ATCC14028 and PB4076 demonstrated that the deletion of the rpoS gene produces a considerable increase in the sunlight sensitivity of S. Typhimurium (Fig. 3). The dose required to reduce viable counts of the mutant to the detection limit allowed for survival of one third of the initial bacterial population of the wild type strain. The parameters of Eq. (1) can be fitted to the experimental data
-1
0
60
120
Irradiation time (min) Fig. 2. Influence of oxygen concentration on survival of strain ATCC14028 exposed to natural sunlight. A bacterial suspension was irradiated in unshielded quartz cells while bubbled with air (open circles) or nitrogen (open triangles). The function obtained by fitting the parameters of Eq. (1) to the experimental results for the sample bubbled with air (solid line), and that obtained by fitting the parameters of the first term in Eq. (1) to the experimental results for the sample bubbled with nitrogen (dashed line) were also plotted. Irradiation start time was 2010-02-2515:25 (UTC), irradiance was 881–894 W m2, and temperature of the samples was 26 °C. The experiment was repeated twice, representative results are shown.
to play a key role to induce the lethal effects observed in ATCC14028.
Table 1 Comparison of the sunlight susceptibility of different S. Typhimurium strains. Strain ATCC14028 PB4076 DA1468
1/q (J m2)a 6
5.75 10 (1.01 106) 1.22 106 (2.62 105) 4.19 106 (9.81 105)
1/k (J m2)a 5
8.43 10 (3.32 105) 2.49 105 (6.95 104) 4.75 105 (1.27 105)
tq (J m2)a 6.60 106 (7.50 105) 3.08 106 (1.30 106) 6.12 106 (2.71 105)
a Parameters of Eq. (1) were fitted to the experimental survival curves. Using the obtained values and solar irradiation data for the wavelength range between 200 and 1100 nm, the fluences required for 1/e (37%) survival from the single-hit (1/q) and multiple-hit (1/k) killing processes, and the fluence equivalent to the quasithreshold dose (tq), were calculated. Mean values from three independent measurements are shown, and the corresponding standard deviations are given in parenthesis.
23
0
0
-2
-2
log (N/N0)
log (N/N0)
O.J. Oppezzo et al. / Journal of Photochemistry and Photobiology B: Biology 102 (2011) 20–25
-4
-4
-6 -6 0
60
120
0
Irradiation time (min) Fig. 4. Influence of solar ultraviolet and oxygen concentration on survival of strain PB4076 exposed to natural sunlight. A bacterial suspension was irradiated in unshielded quartz cells while bubbled with air (open circles) or nitrogen (open triangles), and in quartz cells covered with a polycarbonate sheet (open squares) or with an opaque material (close circles) while bubbled with air. The functions obtained by fitting the parameters of Eq. (1) to the experimental results for the unshielded samples bubbled with air (solid line) and nitrogen (dashed line) were also plotted. Irradiation start time was 2010-03-26-15:30 (UTC), irradiance was 854–874 W m2, and temperature of the samples was 26 °C. The experiment was repeated twice, representative results are shown.
obtained with PB4076. In this strain the initial slope (q) and the additional slope in the final portion of the curve (k) were found increased, and the dose corresponding to the change in the slope (calculated as the quasi-threshold dose) was reduced with respect to the values estimated for the strain ATCC14028 (Table 1). It should be noted that the survival curves for PB4076 could also be modeled using a Weibull type model proposed for convex inactivation curves [25], which can be reduced to the expression of a simple exponential decay elevated to an exponent whose physical meaning is undefined. Since this model is not suitable to describe inactivation of ATCC14028, the rpoS mutation might slightly modify the shape of the survival curve, and a comparison of the numerical values estimated for the parameters of Eq. (1) would not allow a complete description of the effects of this mutation. Similarly to the wild type strain, lethal effects were undetectable in PB4076 when the ultraviolet component was excluded from the incident radiation (Fig. 4). Since both, wild type and mutant bacteria were resistant to the effects of visible light, it may be inferred that RpoS is required to overcome the effects of ultraviolet radiation during sunlight exposures. Nitrogen bubbling provided considerable protection to the mutant and this effect could be observed even at low doses (Fig. 4), suggesting that the increment in lethal effect in the rpoS mutant depends to a great extent on the photodynamic action of the radiation. The same experimental approach was used with a S. Typhimurium strain derived from LT2, carrying the rpoS mutation present in this strain. Survival curves obtained for the strain DA1468 had a shape similar to that of the curve for ATCC14028 (Fig. 3). Fitting the parameters of Eq. (1) to the experimental data the value for k was found increased in DA1468 with respect to that estimated for ATCC14028 (Table 1). When suspensions of DA1468 were shielded against solar ultraviolet or bubbled with nitrogen, the changes observed in the survival curves were identical to those found with ATCC14028 (data not shown). Conclusions about the
60
120
Irradiation time (min) Fig. 5. Effect of rpoS inactivation on survival of strain DA1468 exposed to natural sunlight. Suspensions of strains DA1468 (open pentagons), and DA2245 (open inverted triangles), were irradiated in unshielded quartz cells while bubbled with air. The function obtained by fitting the parameters of Eq. (1) to the experimental results for DA1468 (solid line) and that obtained by fitting the parameters of Eq. (3) to the experimental results for DA2245 (doted line) were also plotted. Irradiation start time was 2010-03-16-15:54 (UTC), irradiance was 860–866 W m2, and temperature of the samples was 25 °C. The experiment was repeated twice, representative results are shown.
importance of solar ultraviolet and the contribution of the photodynamic action to the lethal effects of sunlight on ATCC14028 can be extended to DA1468, suggesting that the increased sensitivity found in these strains is related with the damage induced by reactive oxygen species. To estimate the contribution of systems under RpoS control to the survival of DA1468 exposed to sunlight, the response of this strain was compared with that of its derivative DA2245, which carries an insertion inactivating rpoS. Increased sensitivity found in DA2245 compared to DA1468 demonstrates that RpoS functions related with photoprotection are conserved in strains derived from LT2 (Fig. 5). The shape of the survival curve for DA2245 was very different to that found for PB4076. No shoulder was apparent and the rate of cell death decreased when the irradiation was prolonged producing a tail and suggesting the presence of a bacterial subpopulation with increased resistance. 4. Discussion The strain ATCC14028 exhibited considerable sunlight tolerance, as described by other laboratories [5,6]. However, in contrast with log-linear survival curves previously reported for this strain [5], a complex cell death kinetics was observed. The sunlight response of ATCC14028 was dependent on oxygen concentration in the irradiation medium, and log-linear survival curves were obtained only at low oxygen concentrations. Differences in oxygen availability during the exposures could likely be the reason for the discrepancy between the results presented here and those reported by other researchers. A previous study concerning the influence of an rpoS mutation on the sunlight response of ATCC14028 was focused on bacterial survival in a marine environment [14], and the effects of radiation were assayed under experimental conditions similar to those expected in such an environment. Survival curves were obtained
24
O.J. Oppezzo et al. / Journal of Photochemistry and Photobiology B: Biology 102 (2011) 20–25
excluding solar ultraviolet from the incident radiation, and bacteria were suspended in seawater during irradiations. It is difficult to compare the results obtained under these conditions with those described here, since it has been shown that exposure to seawater increases the sensitivity of Salmonella wild type cells to natural sunlight [26], and that rpoS inactivation reduces the ability of S. enterica to tolerate the stress produced by seawater in the dark [27]. Interestingly, solar ultraviolet is required for the induction of lethal effects in ATCC14028, and RpoS seems to be involved in the defense systems against these effects, resembling the situation described in P. syringae, an organism adapted to a very different environment [12]. When ATCC14028 was irradiated under a nitrogen atmosphere the survival curve seemed linear in a semi-logarithmic plot, and exhibited a slope comparable to the initial slope in curve for cells bubbled with air (Fig. 2). This observation seems compatible with the model formulated in Eq. (1) assuming that the inactivation of targets responsible for cell killing with single-hit single-target kinetics is almost independent of the oxygen concentration in the irradiation medium, but the events responsible for accumulative lethal damage depend on this condition. Recent studies remark the role of oxidative damage to proteins in cell death induced by ultraviolet A radiation in E. coli [28,29]. A lost of viability produced by protein oxidation could exhibit single-hit multiple-target kinetics. If a single-hit were sufficient to inactivate a molecule of the target protein the conditions assumed in this model [23] would be fulfilled, because bacterial cells would contain many molecules of this protein, which would have the same probability of being inactivated, and inactivation of a single protein molecule should not produce cell death. The inactivation of a target protein, eventually a component of the respiratory chain [28], by reactive oxygen species generated during sunlight exposure could explain the cell death with single-hit multiple-target kinetics, the protective effect of nitrogen bubbling and, to some extent, the role of RpoS since in stationary phase this sigma factor controls the expression of genes involved in prevention of oxidative damage [10]. Nevertheless, the effect of the rpoS deletion on the survival curves of S. Typhimurium was not limited to the increase in the slope ascribed to the oxidative damage (Table 1 and Fig. 3), suggesting a more complex role for RpoS in the sunlight response of this bacterium. Whilst the survival curves obtained for PB4076 (Figs. 3 and 4) were comparable to those reported for E. coli rpoS mutants exposed to sunlight [5,14], the response of DA2245 was different (Fig. 5). Since RpoS is expected to be absent in both PB4076 and DA2245, differences in the survival curves of these strains could be attributed to their genetic backgrounds. In E. coli the expression of some genes involved in oxidative stress tolerance is controlled by both RpoS and OxyR, and the lack of one regulatory system can be partially compensated by the activity of the other [30]. If some genes related with sunlight response were expressed independently of RpoS in S. Typhimurium, the presence of different alleles of these genes in PB4076 and DA2245 could account for the differences in the radiation responses of these mutants. In conclusion, lethal effects induced by sunlight in S. Typhimurium depend on solar ultraviolet, and the contribution of the photodynamic action of the radiation to cell death is limited in the wild type strain, but relevant in rpoS mutants. Models used to analyze the action of solar radiation on S. Typhimurium should take into account the complex nature of this action to provide suitable descriptions of the survival curves. Since the genetic background of the rpoS mutant strains seems to modify the consequences of rpoS inactivation, descriptions of the influence of other genes on the sunlight response of S. Typhimurium would be necessary to understand the survival strategies of this organism.
Acknowledgments We thank F.C. Soncini for bacterial strains kindly provided. This work has been supported in part by a grant from Agencia Nacional de Promoción Científica y Tecnológica (PICT 38241). References [1] A.L.H. Gameson, J.R. Saxon, Field studies on effect of daylight on mortality of coliform bacteria, Water Res. 1 (1967) 279–295. [2] C.E. Chamberlin, R. Mitchell, in: R. Mitchell (Ed.), Water Pollution Microbiology, vol. 2, John Wiley and Sons, New York, 1978, pp. 325–348. [3] Y. Rozen, S. Belkin, Survival of enteric bacteria in seawater, FEMS Microbiol. Rev. 25 (2001) 513–519. [4] M. Wegelin, S. Canonica, K. Mechsner, T. Fleischmann, F. Pesaro, A. Metzler, Solar water disinfection: scope of the process and analysis of radiation experiments, J. Water Supply Res. Technol.-Aqua. 43 (1994) 154–169. [5] M. Berney, H.-U. Weilenmann, A. Simonetti, T. Egli, Efficacy of solar disinfection of Escherichia coli, Shigella flexneri, Salmonella Typhimurium and Vibrio cholerae, J. Appl. Microbiol. 101 (2006) 828–836. [6] F. Bosshard, M. Berney, M. Scheifele, H.-U. Weilenmann, T. Egli, Solar disinfection (SODIS) and subsequent dark storage of Salmonella Typhimurium and Shigella flexneri monitored by flow cytometry, Microbiology 155 (2009) 1310–1317. [7] R.O. Fernández, R.A. Pizarro, Lethal effect induced in Pseudomonas aeruginosa exposed to ultraviolet-A radiation, Photochem. Photobiol. 64 (1996) 334–339. [8] O.J. Oppezzo, R.A. Pizarro, Sublethal effects of ultraviolet A radiation on Enterobacter cloacae, J. Photochem. Photobiol. B: Biol. 62 (2001) 158–165. [9] M. Kennedy, R. Villar, D.J. Vugia, T. Rabatsky-Ehr, M.M. Farley, M. Pass, K. Smith, P. Smith, P.R. Cieslak, B. Imhoff, P.M. Griffin, Hospitalization and deaths due to Salmonella infections, FoodNet 1996–1999, Clin. Infect. Dis. 38 (3) (2004) S142–S148. [10] H. Weber, T. Polen, J. Heuveling, V.F. Wendisch, R. Hengee, Genome-wide analysis of the general stress response network in Escherichia coli: rsdependent genes, promoters, and sigma factor selectivity, J. Bacteriol. 187 (2005) 1591–1603. [11] R.W. Tuveson, R.B. Jonas, Genetic control of near-UV (300–400 nm) sensitivity independent of the recA gene in strains of Escherichia coli K12, Photochem. Photobiol. 30 (1979) 667–676. [12] C.D. Miller, W.S. Mortensen, G.U.L. Braga, A.J. Anderson, The rpoS gene in Pseudomonas syringae is important in surviving exposure to the near-UV in sunlight, Curr. Microbiol. 43 (2001) 374–377. [13] S. Zaafrane, K. Maatouk, J.M. Gauthier, A. Bakhrouf, Influence des conditions de culture préalables et de la présence du gène rpoS pour la survie de Salmonella Typhimurium en eau de mer exposée à la lumière solaire, Can. J. Microbiol. 50 (2004) 341–350. [14] M. Berney, H.-U. Weilenmann, J. Ihssen, C. Bassin, T. Egli, Specific growth rate determines the sensitivity of Escherichia coli to thermal, UVA, and solar disinfection, Appl. Environ. Microbiol. 72 (2006) 2586–2593. [15] L. Brown, T. Elliot, Efficient transcription of the RpoS sigma factor in Salmonella Typhimurium requires host factor I, an RNA-binding protein encoded by the hfq gene, J. Bacteriol. 178 (1996) 3763–3770. [16] V. Robbe-Saule, M. Dias Lopes, A. Kolb, F. Norel, Physiological effects of Crl in Salmonella are modulated by rs level and promoter specificity, J. Bacteriol. 189 (2007) 2976–2987. [17] M.R. Wilmes-Riesenberg, J.W. Foster, R. Curtiss, An altered rpoS allele contributes to the avirulence of Salmonella Typhimurium LT2, Infect. Immun. 65 (1997) 203–210. [18] W.E. Swords, B.M. Cannon, W.H. Benjamin, Avirulence of LT2 strains of Salmonella Typhimurium results from a defective rpoS gene, Infect. Immun. 65 (1997) 2451–2453. [19] T. Jarvik, C. Smillie, E.A. Groisman, H. Ochman, Short-term signatures of evolutionary change in the Salmonella enterica serovar Typhimurium 14028 genome, J. Bacteriol. 192 (2010) 560–567. [20] M.L. Cabeza, A. Aguirre, F.C. Soncini, E. García Véscovi, Induction of RpoS degradation by the two-component system regulator RstA in Salmonella enterica, J. Bacteriol. 189 (2007) 7335–7342. [21] O.J. Oppezzo, D.N. Antón, Involvement of cysB and cysE genes in the sensitivity of Salmonella Typhimurium to mecillinam, J. Bacteriol. 177 (1995) 4524–4527. [22] F.C. Fang, S.J. Libby, N.A. Buchmeier, P.C. Loewen, J. Switala, J. Harwood, D.G. Guiney, The alternative r factor KatF (RpoS) regulates Salmonella virulence, Proc. Natl. Acad. Sci. USA 89 (1992) 11978–11982. [23] E.L. Alpen, Radiation Biophysics, Academic Press, San Diego, 1998. [24] A. Eisenstark, in: J.G. Scandalios (Ed.), Advances in Genetics, vol. 26, Academic Press, San Diego, 1989, pp. 99–147. [25] A.H. Geeraerd, V.P. Valdramidis, J.F. Van Impe, GIanFiT, a free tool to assess non-log-linear microbial survivor curves, Int. J. Food Microbiol. 102 (2005) 95– 105. [26] C.M. Davies, L.M. Evison, Sunlight and survival of enteric bacteria in natural waters, J. Appl. Bacteriol. 70 (1991) 265–274. [27] P.M. Munro, G.N. Flatau, R.L. Clément, J.M. Gauthier, Influence of the RpoS (KatF) sigma factor on maintenance of viability and culturability of Escherichia coli and Salmonella Typhimurium in seawater, Appl. Environ. Microbiol. 61 (1995) 1853–1858.
O.J. Oppezzo et al. / Journal of Photochemistry and Photobiology B: Biology 102 (2011) 20–25 [28] F. Bosshard, M. Bucheli, Y. Meur, T. Egli, The respiratory chain is the cell’s Achilles’ heel during UVA inactivation in Escherichia coli, Microbiology 156 (2010) 2006–2015. [29] F. Bosshard, K. Riedel, T. Schneider, C. Geiser, M. Bucheli, T. Egli, Protein oxidation and aggregation in UVA irradiated Escherichia coli cells as signs of
25
accelerated cellular senescence, Environ. Microbiol., in press. doi:10.1111/ j.1462-2920.2010.02268.x. [30] A.B. Ivanova, G.V. Glinsky, A. Eisenstark, Role of rpoS regulon in resistance to oxidative stress and near-UV radiation in DoxyR suppressor mutants of Escherichia coli, Free Radic. Biol. Med. 23 (1997) 627–636.