ISOLATION OF A DEGENERATION-RESISTANT MUTANT OF CLOSTRIDIUM-ACETOBUTYLICUM NCIMB-8052

Unless periodically grown from germinated spores, Clostridium acetobut ylicum tends to degenerate (that is, to spontaneously lose the capacit y both to produce solvents and to develop into spores). To obtain muta nts that are deficient in degeneration, C. acetobutylicum NCIMB 8052 w as mated with Enterococcus faecalis BM4110 harboring transposon Tn1545 . We developed a degeneration resistance assay based on a secondary ef fect of degeneration, the production of toxic levels of acetic and but yric acids. Erythromycin-resistant transconjugant clones were tested i ndividually for longevity by repeated and timely subculturing. One lon g-lived mutant, A10, survived 18 +/- 3 transfers (mean +/- standard de viation; n = 20) before extinction, while the wild type (parental cell s) survived 6.6 +/- 1.5 transfers (n = 11). The three-fold difference in longevity is statistically significant. In a batch culture in a ric h medium, the wild-type cells degenerated within 24 h after inoculatio n with 1% of an overnight culture derived from germinated spores. In c ontrast, A10 cells were able to switch to solventogenesis and to sporu late. In a minimal medium with greater buffering capacity, both cell t ypes produced solvents and spores. Southern blots of EcoRI and HindIII restriction digests of A10 chromosomal DNA (but not parental DNA) sho wed that only one copy of Tn1545 was inserted into the clostridial chr omosome. Our findings are consistent with the hypothesis that there wa s an alteration at a regulatory locus that was effected by the inserti on of the transposon.