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.

Tail deformity induced by coryneform bacteria: Some mutants are really a pain in the ass

Jonathan Hodgkin, Brit Corneliussen

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).