Worm Breeder's Gazette 15(1): 73 (October 1, 1997)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
MRC Laboratory of Molecular Biology Cambridge CB2 2QH, England
On at least two occasions during the years of worm-breeding at MRC-LMB, distinctive morphological variants have arisen spontaneously and been used to found apparently stable mutant lines. These worms have abnormal tail morphology in hermaphrodites, with a pronounced post-anal swelling, especially conspicuous in L4 and adult. This phenotype was designated Dar, for Deformed Anal Region. Two independent Dar lines have been examined in detail. In both cases, the Dar trait behaved in mapping crosses as a dominant mutation, not closely linked to any of the autosomal clusters. In further crosses, the trait was found to segregate in a non-Mendelian pattern, and it was eventually realized that the Dar phenotype is not genetically determined. Instead, it is caused by a bacterial infection, although there is no obvious contamination in the E. coli lawns used to propagate Dar lines. Three pieces of evidence demonstrated the presence of an infection. First, Dar strains can be completely cured by alkaline hypochlorite sterilization. Second, culturing Dar hermaphrodites marked with dpy-12 together with wild-type N2 hermaphrodites leads to the appearance of non-Dpy Dar hermaphrodites, after a few days. Third, close examination of the post-anal swelling under Nomarski optics revealed the presence of a small patch of rod-shaped non-coliform bacteria, tightly adhering to the cuticle over the deformed region. It proved possible to grow these bacteria in the absence of worms. Single Dar worms were starved on unseeded NGM agar for several hours, and then picked to TYE plates at 37 degrees C. The worms crawl for some time before expiring, leaving behind (after incubation) a trail of a few large E. coli colonies (OP50) and a larger number of small, slow-growing colonies. When these bacteria are grown up and added back in trace amounts to E. coli lawns with N2 worms, they are capable of generating the Dar phenotype in N2 hermaphrodites, thus satisfying Koch's postulates. Three distinct Dar-producing bacterial strains have now been isolated, and given the names CBX101, CBX102 and CBX103. The Clinical Microbiology Laboratory at Addenbrooke's Hospital, Cambridge kindly identified all three as gram-positive coryneform bacteria. Coryneforms are a large, taxonomically complex group of soil bacteria, so further investigation will be required to identify them more specifically. The Dar response, with substantial post-anal swelling and deformation, appears to be a sort of inflammatory response by the worms to the adherent bacteria. From these initial observations, it is not clear whether it benefits the worms or the bacteria. One possibility is that the swelling increases the likelihood of the bacteria being scraped off the cuticle. From the bacterial point of view, adherence to posterior cuticle is likely to be an effective survival strategy. C. elegans is able to eat and digest these bacteria, because worms will grow and reproduce on lawns consisting only of CBX102. Therefore, the post-anal patch may be the safest and most nutritious place to be, in a culture of starving nematodes. Although the bacteria grow much more slowly than E. coli, they survive better on starved plates. This was demonstrated by adding worms to a lawn consisting of OP50 and traces of CBX102 (starting ratio 1600:1). When the worms had eaten the lawn and begun to starve, the remaining bacterial population was sampled by rinsing the plate with buffer, and plating this on nutrient media. OP50 colonies were found to be much less numerous than CBX102 colonies (final ratio about 1: 100).