An alternative interpretation for tailing in survival curves for bacteria exposed to germicidal radiation.

It has been proposed that transient and reversible phenotypic changes could modify the response of bacteria to germicidal radiation, eventually leading to tailing in the survival curves. If this were the case, changes in susceptibility to radiation would reflect variations in gene expression and should only occur in cells in which gene expression is active. To obtain experimental evidence supporting the involvement of phenotypic changes in the origin of tailing, we studied changes in the susceptibility to radiation of cells able to survive high fluences, using split irradiations. Stationary phase cells of Enterobacter cloacae and Deinococcus radiodurans, in which gene expression is active, and spores of Bacillus subtilis, which are dormant cells without active gene expression, were used as microbial models. While cells of E. cloacae and D. radiodurans became susceptible after surviving exposures to high fluences, tolerant spores exhibited unchanged response to radiation. The results can be interpreted assuming that noise in gene expression modifies bacterial susceptibility to radiation, and tailing is the result of intrinsic phenomena of bacterial physiology rather than a technical artifact. For either theoretical or practical purposes, deviations from simple exponential decay kinetics should be considered in estimations of the effects of germicidal radiation at high fluences.

Protective Effects of Halite to Vacuum and Vacuum-Ultraviolet Radiation: A Potential Scenario During a Young Sun Superflare.

Halite (NaCl mineral) has exhibited the potential to preserve microorganisms for millions of years on Earth. This mineral was also identified on Mars and in meteorites. In this study, we investigated the potential of halite crystals to protect microbial life-forms on the surface of an airless body (e.g., meteorite), for instance, during a lithopanspermia process (interplanetary travel step) in the early Solar System. To investigate the effect of the radiation of the young Sun on microorganisms, we performed extensive simulation experiments by employing a synchrotron facility. We focused on two exposure conditions: vacuum (low Earth orbit, 10-4 Pa) and vacuum-ultraviolet (VUV) radiation (range 57.6-124 nm, flux 7.14 W/m2), with the latter representing an extreme scenario with high VUV fluxes comparable to the amount of radiation of a stellar superflare from the young Sun. The stellar VUV parameters were estimated by using the very well-studied solar analog of the young Sun, κ1 Cet. To evaluate the protective effects of halite, we entrapped a halophilic archaeon (Haloferax volcanii) and a non-halophilic bacterium (Deinococcus radiodurans) in laboratory-grown halite. Control groups were cells entrapped in salt crystals (mixtures of different salts and NaCl) and non-trapped (naked) cells, respectively. All groups were exposed either to vacuum alone or to vacuum plus VUV. Our results demonstrate that halite can serve as protection against vacuum and VUV radiation, regardless of the type of microorganism. In addition, we found that the protection is higher than provided by crystals obtained from mixtures of salts. This extends the protective effects of halite documented in previous studies and reinforces the possibility to consider the crystals of this mineral as potential preservation structures in airless bodies or as vehicles for the interplanetary transfer of microorganisms.

Survival of Pseudomonas aeruginosa exposed to sunlight resembles the phenom of persistence.

During exposure of Pseudomonas aeruginosa stationary phase cells to natural solar radiation, a reduction in the rate of loss of bacterial viability was observed when survival fractions were lower than 1/10,000. This reduction was independent of the growth medium used and of the initial bacterial concentration, and was also observed when irradiation was performed with artificial UVA radiation (365nm, 47Wm(-2)). These results indicate the presence of a small bacterial subpopulation with increased tolerance to radiation. Such a tolerance is non-heritable, since survival curves comparable to those of the parental strain were obtained from survivors to long-term exposure to radiation. The radiation response described here resembles the phenomenon called persistence, which consists of the presence of a small subpopulation of slow-growing cells which are able to survive antibiotic treatment within a susceptible bacterial population. The condition of persister cells is acquired via a reversible switch and involves active defense systems towards oxidative stress. Persistence is probably responsible for biphasic responses of bacteria to several stress conditions, one of which may be exposure to sunlight. The models currently used to analyze the lethal action of sunlight overestimate the effect of high-dose irradiation. These models could be improved by including the potential formation of persister cells.

Anr, the anaerobic global regulator, modulates the redox state and oxidative stress resistance in Pseudomonas extremaustralis.

The role of Anr in oxidative stress resistance was investigated in Pseudomonas extremaustralis, a polyhydroxybutyrate-producing Antarctic bacterium. The absence of Anr caused increased sensitivity to hydrogen peroxide under low oxygen tension. This phenomenon was associated with a decrease in the redox ratio, higher oxygen consumption and higher reactive oxygen species production. Physiological responses of the mutant to the oxidized state included an increase in NADP(H) content, catalase activity and exopolysaccharide production. The wild-type strain showed a sharp decrease in the reduced thiol pool when exposed to hydrogen peroxide, not observed in the mutant strain. In silico analysis of the genome sequence of P. extremaustralis revealed putative Anr binding sites upstream from genes related to oxidative stress. Genes encoding several chaperones and cold shock proteins, a glutathione synthase, a sulfate transporter and a thiol peroxidase were identified as potential targets for Anr regulation. Our results suggest a novel role for Anr in oxidative stress resistance and in redox balance maintenance under conditions of restricted oxygen supply.

Contribution of UVB radiation to bacterial inactivation by natural sunlight.

The contribution of different components of sunlight to the lethal action exerted by this radiation on bacteria was studied using Pseudomonas aeruginosa ATCC27853 as a model organism. When solar UVB was excluded from the incident radiation by filtering it through a naphthalene solution (cut off 327 nm), significant modifications were observed in the cell-death kinetics. These modifications were comparable to those expected for a reduction of 27-32% in the dose rate, according to the model used in the analysis of the survival curves, and were also observed when the effects of sunlight filtered through polyethylene terephthalate (cut off 331 nm) or polystyrene (cut off 298 nm) were compared. Viability of P. aeruginosa remained almost unchanged when the incident radiation was filtered through a sodium nitrite solution (cut off 406 nm) in order to exclude the UVA and UVB components of sunlight. Nevertheless, a delay in colony formation was detected in bacteria treated in this way, suggesting that a non-lethal effect was exerted by visible light. The results are not consistent with a generally accepted notion which attributes the lethal action of sunlight to the radiation with wavelengths above 320 nm. The characterization of UVB contribution to the lethal effect of sunlight on bacteria is relevant for understanding of the mechanism of cell death, and for improvement of dosimetry techniques and irradiation procedures.

The relationship between carotenoids and sunlight response in members of the family Micrococcaceae.

The aim of this study was to compare the photoprotective effect of carotenoids in phylogentically related bacteria, which synthesize structurally different pigments. Two organisms were isolated from the same environment. Their 16S rDNA sequences and phenotypic characteristics identified them as members of the family Micrococcaceae. Reverse phase HPLC and absorption spectroscopy revealed that one of them, designated RMB40, synthesized 3 carotenoids with 9 conjugated double bonds, whilst the other, designated RMB42, synthesized a single and more hydrophobic pigment carrying 11 conjugated double bonds. Survival curves were obtained during sunlight exposure for both organisms and for carotenoid deficient mutants derived from them. Increased sunlight sensitivity was found in the carotenoidless mutant derived from RMB42. In contrast, pigment depletion had no appreciable effect on the sunlight response of RMB40. It is concluded that the structure of bacterial carotenoid probably exert an important influence on the effectiveness of these compounds to provide photoprotection in vivo.

Influence of rpoS mutations on the response of Salmonella enterica serovar Typhimurium to solar radiation.

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.

Inhibition of sulfur incorporation to transfer RNA by ultraviolet-A radiation in Escherichia coli.

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 m(-2)) imparted at a low fluence rate (41 W m(-2)). 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-(4'-pyrimidin 2'-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.

Transient reduction in the tRNA 4-thiouridine content induced by ultraviolet A during post-irradiation growth in Enterobacter cloacae.

The influence of previous exposure to ultraviolet-A radiation (UVA) was studied on the susceptibility of Enterobacter cloacae to undergo the growth delay effect. Comparison of growth curves corresponding to irradiated and control cells showed that a previous treatment with UVA almost abolished the growth delay effect. UV absorption spectra of tRNA, and reverse phase HPLC analysis of hydrolysed tRNA, demonstrated a low content of 4-thiouridine in E. cloacae cells grown after UVA exposure at low doses. Since 4-thiouridine is the UVA target responsible for initiation of growth delay, this observation explained the influence of previous exposure to UVA on the susceptibility of this organism to undergo growth delay. A similar but weaker alteration was found when Escherichia coli was assayed. The results suggest that, in addition to cross-linking with cytidine residues, the content of 4-thiouridine in tRNA may be modified by UVA by an unknown mechanism.