Induced Quantitative Variation for Penicillin Titre in Clonal Populations of Aspergillus nidulans

Abstract

The variation in penicillin titre within populations of cultures of Aspergillus nidulans derived from untreated conidia and from conidia treated with ethyl methanesulphonate (EMS), near-ultraviolet light in the presence of 8-methoxypsoralen (8MOP) or N-methyl-N'-nitro-N-nitrosoguanidine (NTG), each at several dose levels, was determined. Both mutagentreated and untreated populations showed a continuous distribution of pencillin titres. The population mean titre of the mutagenized populations was decreased and the range of titres was increased relative to those of the control populations. No differences between sister cultures could be detected in three untreated populations, but nine out of ten populations derived from mutagenized conidia showed significant variation for penicillin titre. In general the magnitude of this induced variation increased with increasing dosage of the mutagen. Comparisons at fixed survival levels indicate that 8MOP mutagenesis is less effective for the induction of variation in penicillin titre than EMS or NTG mutagenesis. A statistical procedure was adopted to classify the survivors as unchanged cultures ('0'), titre-increasing mutants ('+') or titre-decreasing mutants ("-"). The frequency of both '+' and '-' mutants increased following mutagenesis, with NTG being the most active of the three mutagens. Over all treatments, these two mutant classes were recovered with equal frequency. The frequency of "+" mutants was largely independent of mutagen dose, within the ranges used, and moderate treatments (around 10% survival) gave as high or higher frequencies than more extreme doses. All three mutagens, and in particular NTG, produced morphological mutants. These contained an increased frequency of titre-decreasing mutants, but increases in titre appeared to be independent of changes in colony morphology. Estimates based on the observed frequencies of penicillin titre mutants suggest that several hundred genes are potentially capable of affecting this continuous variable.

Recurrent Mutation and Selection for Increased Penicillin Titre in Aspergillus nidulans

“…Preliminary experiments designed to facilitate the choice of mutagen and mutation conditions for this strain development programme have been described previously (Simpson & Caten, 1979). Three mutagens -ethyl methanesulphonate (EMS), near-ultraviolet light in the presence of 8-methoxypsoralen (8MOP) and N-methyl-N'-nitro-N-nitrosoguanidine (NTG) -were used in these preliminary experiments.…”

“…This combination of mutagens has the added advantage that in Eschericlzia coli at least they act through different mutational pathways, 8MOP being dependent upon a functional exrA+/recA+ system while EMS is not (Bridges, 1976). Studies of the dose-response relationship for the production of titre-increasing mutants by 8MOP and EMS suggested that mutant frequency was largely independent of dose over the range giving 28 to 404, survival but declined at survivals of less than I (Simpson & Caten, 1979).…”

“…However, examination of the relationship between changes in penicillin titre and colony morphology for the strains of A . nidulans indicated that there was no advantage to be gained from such pre-selection (Simpson & Caten, 1979). Accordingly, both mutagens were used at doses and under conditions previously shown to give 10% survival and strains were sampled at random from the survivors for yield testing.…”

“…Although small, samples of this size should include one or more titre-increasing mutants since the mutagenic treatments used produce such mutants at frequencies of 5 O or more (Simpson & Caten, 1979). Regarding (2) and (3), the usual procedure in strain development programmes has been to carry forward to the next cycle of mutagenesis only the single highest-yielding strain from the population screened (i.e.…”

“…Since the frequency of titre-increasing mutants in the present system was high ( > 5 % ;Simpson & Caten, 1979) and the testing errors low (coefficient of variation 12.6y0), it was decided to use a single-stage screen together with a selection intensity of 5 % , i.e. N2 = 5.…”