Toxicity of Irradiated Media for Xenorhabdus spp

Abstract

Bacterial isolates of the genus Xenorhabdus were shown to be extremely sensitive to photoproducts produced in a number of common media irradiated by fluorescent light. Two forms of toxic oxygen, hydrogen peroxide and superoxide radical, were produced in the media upon exposure to fluorescent light. The addition of pyruvate or catalase to the irradiated media eliminated the toxicity. The poor plating efficiencies previously reported for Xenorhabdus spp. are likely due to the uncontrolled exposure of media to ambient lighting. This study was prompted by the difficulties we and others have reported in obtaining quantitative data on Xenorhabdus viability because of problems of poor plating efficiency (15, 29, 37). Recently, we have observed that the plating efficiency of several Xenorhabdus strains in our laboratory was greatly reduced on plates exposed to ambient fluorescent light. We report here the extreme sensitivity of Xenorhabdus spp. to a variety of media irradiated with fluorescent light and describe various parameters, including protective agents, of the phenomenon. Bacteria of the genus Xenorhabdus form a complex symbiotic association with the entomogenous nematodes of the families Steinernematidae and Heterorhabditidae (29). In natural conditions, the nematodes and Xenorhabdus act cooperatively to kill insects (9, 10, 15, 29), and each depends on the other for its survival (1, 15, 29). Because a large number of insect species are susceptible to Xenorhabdus-nematode infection, the association has potential for the biological control of insect pests (3). The production of toxic photoproducts in bacterial media by near-UV (NUV) visible light has been reported by a number of workers (4, 6, 16, 18, 19, 31, 35, 39). Although the source, nature, and mechanism of formation of toxic photoproducts produced in these media remain in dispute, H202 is reported to be responsible for much of the toxic activity (4, 6, 14, 16, 19, 26, 31, 39). H202 formation has been shown to occur by the action of visible light on tryptophan (17, 26, 39) and on riboflavin and its derivatives (18, 21). Furthermore, photodynamic action of a variety of biological chromophores, including quinones, pteridines, NAD, thiouracil, pyridoxal phosphate, cytochromes, prophyrins, riboflavin, flavin adenine dinucleotide, and flavoproteins has been shown to produce other oxygen species (e.g., hydroxyl radical [OH-], superoxide radical [02' ], and singlet oxygen [1021) which are toxic to bacteria (14, 21-23, 33). The various Xenorhabdus species and strains used, the culture conditions, and the medium preparation were as previously described (20, 37). All media were kept in the dark except for experimental irradiation. H202 was purchased from Fisher Scientific Co., Pittsburgh, Pa. All other chemicals were purchased from Sigma Chemical Co., St.

The global regulator Lrp contributes to mutualism, pathogenesis and phenotypic variation in the bacterium Xenorhabdus nematophila

“…Liquid bacterial cultures were grown in LB broth (Miller, 1972) at 30°C. Liquid media used with X. nematophila strains were stored in the dark and solid media were solidified with 2% agar and supplemented with 0.1% sodium pyruvate (Xu and Hurlbert, 1990). Defined agar plates were made as previously described (Orchard and Goodrich-Blair, 2004).…”

Clonal variation in Xenorhabdus nematophila virulence and suppression of Manduca sexta immunity

“…Our standard culture media for X. nematophila contain 0.1% pyruvate to counteract photoproducts detrimental to X. nematophila growth (Xu and Hurlbert, 1990). On such plates, we discerned no visible morphological differences between vmo and non-vmo populations (data not shown).…”

“…Bacterial strains and plasmids used in this study are listed in Table S2. All bacterial cultures were grown in LB broth (Miller, 1972) that was either stored in the dark or supplemented with 0.1% sodium pyruvate (Xu and Hurlbert, 1990) except when monitoring translucence, in which case untreated LB was used. Tobacco hornworm M. sexta insects obtained from the North Carolina State University Entomology Insectary (Raleigh, North Carolina) were used in all experiments except those presented in Figs 1A and 2, for which M. sexta were obtained from W. Goodman (University of Wisconsin-Madison).…”

Identification of Xenorhabdus nematophila genes required for mutualistic colonization of Steinernema carpocapsae nematodes

“…M. sexta (tobacco hornworm) insect eggs were obtained from W. Goodman (University of Wisconsin-Madison) and were reared as described by Vivas and Goodrich-Blair (2001). Media were supplemented with Na-pyruvate (0.1%) (Xu and Hurlbert, 1990), kanamycin (Km, 50 µg ml −1 ), rifampicin (Rif, 100 µg ml −1 ) or chloramphenicol (Cm, 15 µg ml −1 ), where appropriate.…”