The rapid isolation of mutants of some Gram-positive bacteria

Abstract

1. INTRODUCTION Mutant analysis facilitates, in several ways, the study of cellular processes at the molecular level. With the proper series of mutants, for example, it is possible to define the enzymatic steps in a biochemical pathway. With a multiply-marked strain it is possible to map the structural gene for a protein of particular interest. Knowledge of the location of a marker simplifies the task of prepar- ing strains containing different combinations of mutations which can be used to analyse the regula- tion of particular biochemical pathways. A number of problems have been encountered when attempts have been made to isolate, for the first time, a stock of mutants of a hitherto ne- glected organism. First, well-tried methods for ob- taining mutants of several bacterial species are lacking. Second, since two organisms rarely will respond in precisely the same way to a mutagenic treatment, or a mutant screening programme, it of tern happens that a published procedure has to be modified to yield optimal results. Finally, pro- vision must be made for segregation of pure mutant cells before cloning so as not to isolate strains all bearing exactly the same mutation. Thus isolation of independent mutants requires independent mutagenic treatments. In this communication we described a method for isolating at high frequency independent mutants of a number of Gram- positive bacteria. The method was originally devel- oped for use with an Arthrobacter sp. and appears to work best with this and other coryneform bacteria. 2. MATERIALS AND METHODS All the bacteria used were from the culture collections maintained at the University of Warwick or the Centre for Applied Microbiologi- cal Research. Arthrobacter P1 and coryneform D7F have been described elsewhere [1,2]. The complex media used were TSBA (for Bacil- lus sp.) and Oxoid nutrient agar (all other strains). TSBA contained, per litre, 17 g Oxoid tryptone, 3 g Oxoid soya peptone, 5 g NaC1, 2.5 g KzHPO 4, 2.5 g glucose and 15 g agar; the pH was adjusted to 7.3 with 2 N HCI prior to sterilization. The minimal medium used was that of Spizizen [3] except for Arthrobacter, when the medium of Levering et al. [1] was used. Minimal media were supplemented with amino acids as required. Nutri- tional pool plates were prepared as described by Clowes and Hayes [4]. For mutagenesis using UV light, cells were grown in complex media and used while still in the logarithmic phase of growth. Details of the irradia- tion procedure are given in the text.