Sublethal effects of ultraviolet A radiation on Enterobacter cloacae

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Abstract

We report the sublethal effects of ultraviolet A (UVA) on Enterobacter cloacae in comparison with those produced in Escherichia coli. UVA-induced sublethal effects were investigated in either bacterial membrane and at tRNA level. Limited dependence on oxygen concentration for photoinduced inhibition of biochemical membrane functions and low levels of oxidative damage during the irradiation period were found in En. cloacae. On the other hand, ultraviolet spectroscopy and reversed-phase HPLC analysis of hydrolysed tRNA showed that radioinduced damage to tRNA is similar in En. cloacae and E. coli. Nevertheless, growth delay induced by UVA in En. cloacae was shorter than that found in E. coli submitted to the same experimental conditions. A limited post-irradiation ppGpp accumulation and the absence of any influence of the membrane damage on the growth delay extent seem to be responsible for the shortness of this effect in En. cloacae. Most of the differences between En. cloacae and E. coli could be attributed to an increased ability of En. cloacae to overcome oxidative stress during UVA exposure.

Introduction

Deleterious effects of solar radiation on bacteria have long been recognised [1], [2]. The ultraviolet component of solar radiation reaching the ground, composed by ultraviolet A (320–400 nm) (UVA) and traces of ultraviolet B (290–320 nm), is one of the most common stressing agents confronted by bacteria in their environment.

The effects induced by ultraviolet A on bacterial cells have been extensively investigated in Escherichia coli and it has been established that exposure to ultraviolet A may cause lethal and mutagenic damage to this organism [3], [4]. Low doses of ultraviolet A induce many physiological alterations, mainly the well-known radioinduced growth inhibition. This phenomenon was attributed to photodamaged tRNAs that lose their charge capacity both slowing down protein synthesis and triggering the stringent response with an increase in the levels of guanosine tetra- and pentaphosphates [4]. Furthermore, oxidative disturbance of bacterial membranes induced by ultraviolet A contributes to enlarge the growth inhibition in Escherichia coli [5], [6].

Similar effects of ultraviolet A were reported in Salmonella thyphimurium [7], [8] and Bacillus subtilis [9]. Nevertheless, in recent studies the response of Pseudomonas aeruginosa to ultraviolet A was found to be very different from that observed in the afore mentioned Gram negative bacilli [10], [11], suggesting that important differences in ultraviolet A susceptibility probably exist among bacterial species. Since little is known about the UVA action on other bacterial species, the aim of the present study was to investigate the sublethal effects of UVA on a common soil and aquatic Gram negative rod, Enterobacter cloacae.

The results reported herein demonstrate that Enterobacter cloacae is more resistant to UVA radiation than the other Gram negative bacilli studied. Radioinduced alterations were investigated and reasons for resistance to sublethal effects are reported.

Section snippets

Bacterial strains and growth conditions

Enterobacter cloacae (29C/M-A4 K. F. Mayer UCL Berkeley), Escherichia coli K12 (ATCC 15153), and Pseudomonas aeruginosa (ATCC 15153) were used throughout. Bacterial cells were incubated at 37°C with vigorous shaking in Luria–Bertani (LB) broth (10 g tryptone, 5 g yeast extract and 5 g NaCl/l of distilled water) [12] or in MOPS medium [13] supplemented with 0.4% glucose. When required, LB medium was made solid by the addition of 15 g/l agar. Bacterial growth was monitored by measuring the

UVA-induced growth delay in En. cloacae

One of the most studied effects of the sublethal UVA exposure of bacterial cells is a transient block in the cell growth. Previously, it was reported that the exposure of E. coli cells suspension to 120 kJ m−2 of UVA results in a growth delay lasting approximately 90 min [5]. When En. cloacae suspensions were treated in the same experimental conditions, the UVA-induced growth delay effect was observed. In fact, a dose dependent displacement of the growth curve was found (Fig. 1) with no changes

Discussion

Among the information available concerning the biological effects of UVA on bacteria, few data about differences in the sensitivity between Gram negative bacilli are disposable. The aim of the present study was to investigate the UVA response of a common soil and aquatic microorganism, Enterobacter cloacae. The results show that the ability En. cloacae to overcome the UVA deleterious effects is greater than that found in other Gram negatives.

Alterations on membrane functions were found in E.

Acknowledgements

We thank Dr. R.O. Fernández for helping to develop HPLC procedures, Dr. A.E. Schwint for helping to correct the manuscript, and Mr. Jorge A. Ibáñez for technical assistance.

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