Department of Molecular Biology, University of Gdask, K
adki 24, 80-822 Gda
sk, Poland1
Marine Biology Center, Polish Academy of Sciences, w. Wojciecha 5, 81-347 Gdynia, Poland2
Author for correspondence: Grzegorz Wegrzyn. Tel: +48 58 346 3014. Fax: +48 58 301 0072. e-mail: wegrzyn{at}biotech.univ.gda.pl
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ABSTRACT |
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Keywords: bioluminescence, Vibrio harveyi lux genes, DNA repair, photoreactivation, OS response
We would like to dedicate this paper to the memory of Karol Taylor, who introduced V. harveyi projects to our laboratories.
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INTRODUCTION |
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There are several known bacterial species that are able to emit light (Meighen, 1994 ). In fact, light-emitting bacteria are the most abundant and widespread of the luminescent organisms found in marine, freshwater and terrestrial habitats. The process of luminescence is found in symbiotic, saprophytic, parasitic, as well as in free-living bacteria (for a review see Meighen, 1994
). The ecological benefit for a fish or squid living in symbiotic association with luminescent bacteria has been established (Morin et al., 1975
; Nealson & Hastings, 1979
). The host organism can use light emitted by bacteria for attraction of prey, escape from predators or intraspecies communication (Morin et al., 1975
; Bassler & Silverman, 1995
). However, it is not understood what specific benefit free-living or symbiotic bacteria derive from producing light (compare Bassler & Silverman, 1995
). On the other hand, it seems obvious that luminescence must have a positive selective value since as much as several per cent of the bacterial cell energy is consumed by this process (Makemson, 1986
; Bassler & Silverman, 1995
). Some speculations on the potential biochemical role of bacterial luminescence were reported (e.g. that the light-emitting system could function as an alternative pathway for electron flow) (Nealson & Hastings, 1979
), but they have never been verified experimentally.
In the course of our studies on the free-living bioluminescent marine bacterium Vibrio harveyi, we have isolated many random mutants. Among these, several mutants were very sensitive to UV irradiation. Surprisingly, we found that most of these mutants had also lost the ability to emit light. Further studies, described in this article, led us to propose that production of internal light ensuring effective DNA repair, most probably by photoreactivation, may be at least one of the biological functions of bacterial luminescence.
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METHODS |
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Transposon mutagenesis.
Transposon mutagenesis of V. harveyi was performed using a combination of previously described modified transposon mutagenesis (MacKenzie et al., 1995 ) and P1 transduction (Rosner, 1972
) procedures.
UV sensitivity assays.
V. harveyi strains were cultivated in BOSS medium, centrifuged and resuspended in 3% NaCl. Following UV irradiation of 1x108 cells, bacteria were incubated in BOSS medium in the dark or under a white fluorescent lamp for 2 h, and then titrated on BOSS plates (the plates were incubated overnight in the dark). An analogous procedure was employed for E. coli strains, but LB medium was used instead of BOSS, and 0·9% NaCl was used instead of 3% NaCl. In the agar plate test, bacteria were streaked across the plate, and sectors of the plate were irradiated with different UV doses. The plate was incubated overnight in the dark, and growth inhibition was estimated.
Measurement of bacterial luminescence.
V. harveyi strains were grown to high cell density (1x107 cells ml-1) in minimal medium 3 containing 3% NaCl. Then, the cultures were diluted 10000-fold in fresh minimal medium 3 and cultivated for 5 h in order to minimize luminescence. Bacteria were irradiated with different doses of UV and incubation was continued for the indicated time periods. Luminescence was monitored at each stage of cultivation in a scintillation counter using chemiluminescence mode as described previously (Bassler et al., 1994 ). The relative light units were calculated as counts min-1 ml-1 per cell. Luminescence of E. coli strains was measured by the method described above, but appropriate medium was used and the cultures were not diluted.
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RESULTS AND DISCUSSION |
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One possible interpretation of the unexpectedly large proportion of non-luminescent mutants among UV-sensitive bacteria was that V. harveyi cells unable to emit light may be defective in the repair of DNA lesions caused by UV light. To test this hypothesis, we investigated the survival of UV-irradiated V. harveyi cells which were subsequently grown in the dark or in the presence of external light (under a white fluorescent lamp). This type of experiment has previously been used to investigate the efficiency of photoreactivation, a process of DNA repair by photolyase (Kato et al., 1997 ). We found that UV-mediated killing of wild-type (luminescent) V. harveyi cells was somewhat more effective when bacteria were cultivated in the dark following irradiation compared to cultivation in the presence of external light (Fig. 1a
). However, significantly less survival of cells was observed when UV-irradiated luxA or luxB (non-luminescent) mutants were cultivated in the dark (Fig. 1b
, c
). The luxA and luxB genes encode the two subunits of luciferase. To test whether the observed defects in DNA repair were caused by the loss of luminescence or the loss of the luciferase enzyme itself, we investigated the UV-sensitivity of the luxD mutant. The luxD gene encodes the acetyltransferase enzyme producing fatty acids for the luminescence reaction, thus luxD mutants are non-luminescent even in the presence of wild-type luciferase. We found that the luxD mutant was significantly more sensitive to UV irradiation when subsequent cultivation was performed in the dark than in the presence of external light, similarly to luxA and luxB mutants (results not shown). These results led us to propose that luminescence may serve as an internal source of light which is used in a photoreactivation-type reaction when bacteria grow in the dark.
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There is one more question concerning this hypothesis about a biological role for bacterial luminescence. It is well established that luminescent bacteria (including V. harveyi) emit light efficiently only when they are at high cell density. This regulation is known as quorum sensing (for a review see Swift et al., 1998 ). If V. harveyi were able to emit light only at high cell density irrespective of other environmental conditions, our hypothesis would seem rather unlikely as mechanisms ensuring efficient DNA repair should also operate at low cell density. To test if UV irradiation can induce luminescence of V. harveyi cells growing at low density, we have diluted a high-density bacterial culture 10000-fold, continued cultivation until the luminescence was negligible, and then irradiated the bacteria with UV light. Bioluminescence was monitored at each stage of the experiment in a scintillation counter using chemiluminescence mode. We found that while light emission by V. harveyi cells growing at low density was very low relative to high cell density conditions or immediately after dilution, UV irradiation of cells at low density caused transient but efficient induction of light emission (Fig. 3
). A dose-response correlation between UV irradiation and luminescence was observed (Fig. 4
). Most probably, stimulation of luminescence was caused by inactivation of the LexA repressor and subsequent induction of the SOS response. To test this hypothesis, we investigated the efficiency of luminescence of E. coli lexA+ and lexA3 (unable to induce the SOS response due to the presence of the uncleavable form of LexA) cells bearing the V. harveyi lux genes on plasmids. UV irradiation of lexA+ cells caused a transient but about a 100-fold increase in luminescence, whereas these conditions had little effect on light emission by the lexA3 mutant (Fig. 5
). Some dose-response correlation between UV irradiation and stimulation of luminescence of lexA+ cells was observed (Fig. 6
). These results support the proposal that V. harveyi genes responsible for the bioluminescence phenotype are under negative control of the SOS response regulator, and that effective luminescence of V. harveyi is possible at low cell density under conditions causing DNA damage.
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ACKNOWLEDGEMENTS |
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Received 20 July 1999;
revised 8 October 1999;
accepted 26 October 1999.