Molecular cloning and characterization of the lux genes from the secondary form of Xenorhabdus luminescens, K122.

Xenorhahdus lurninescens is a luminous bacterium which is symbiotic with the entomopathogenic nematode Hererorhabdiris. The bacteria play an important role in pathogenesis of insect larvae. Once the nematode enters the larvae, Xennrhabdus spp are released from the gut of the nematode into the hemocoel of the insect. Here both the bacteria and the nematodes proliferate and kill the insect within 48 hours 11, 21. It has been reported that bacteria-free nematodes or nematodes with symbiotic bacteria other than Xenorhabdus are often greatly curtailed in their growth and reproduction inside insect hosts [3,4]. Phase variation is a common feature of Xenorhabdus spp. The primary form of Xennrhabdus spp occurs naturally in the infective stage nematode. Cultures of the primary form of Xenorhabdus spp give rise to secondary forms after prolonged static incubation in broth media. The primary form of Xenorhabdus spp provides better growth conditions for the nematodes than does the secondary form 131. Unlike the primary form of Xenorhabdus spp, the secondary form is deficient in pigment and protease and lipase activities, and is about 100 fold less luminous than the primary form in vivo [4]. The primary form symbiont is of major importance in the nematodebacteria association. Although the two forms of Xenorhabdus display no significant difference in their pathogenicity to the Galleria larvael31, the form change can result in a dramatic yield decrease during mass production of the nematodes in vitro[5]. Reversion from the secondary form of XenorhaMus back to the primary form has been demonstrated for some Xenorhabdus spp but not for X lurninescens 161. The genetic mechanism of conversion from primary form to secondary form is not understood. All of the observations are consistent with the view that the secondary variants arise not from random mutations, but from genetic events[7]. The bioluminescence which is 100 times stronger in the primary form than in the secondary form of X. luminescens requires the expression of the lux genes. We have cloned these genes as a first step towards reaching an understanding of the genetic mechanism of phase switching. In order to clone the lux genes, a genomic DNA bank has been constructed from X. lurninescens, K122. Chromosomal DNA isolated from the secondary form was partialy digested with 0.02 units of enzyme Sau3A at 37 "C for 30 minutes. The partially digested DNA fragments were then cloned into the BamHI site of plasmid pUC19, and transformed into the competent cells of E. cnli Tg-1. Two clones, pL1217 and pL1219, were selected out from the DNA bank under dark conditions. Clones pL1217 and pL1219 have 10.5 kb and 17 kb inserts repectively, and must contain the 51ux genes which encode the two subunits of luciferase and the enzymes of the fatty acid reductase complex in order to generate luminescence in E. coli.. To further study the 10.5 kb insert from pL1217, physical mapping of the genes has been done with 11 different enzymes (Fig. 1). The results of mapping showed that the restriction enzyme sites in genes from K122 were slightly different from the sites in genes of other X luminescens strains (8.91. For example, the BamHI site was found in the genes from the K122 strain, but not in the genes from the Hm strain181,