Downloaded from www.microbiologyresearch.org by IP: 107.21.181.127 On: Thu, 09 Feb 2017 10:41:05 Journal of General Microbiology (I 983), 129, 209 1-2 10 1. Printed in Great Britain 209 1 Variety of Sporulation Phenotypes Resulting from Mutations in a Single Regulatory Locus, spoIZA , in BaciIIus subtilis By J. ERRINGTON AND J. MANDELSTAM* Microbiology Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3Q U, U. K. (Received 31 December 1982; revised 14 February 1983) Closely linked mutations in either of the two putative genes of the sporulation locus spoIIA can affect, in quite diverse ways, spore incidence, the production of alkaline phosphatase and DNAase, and the stability of the cells in sporulation medium. It is concluded that the locus has a regulatory function affecting the activation or induction of at least two, and possibly more, sporulation-associated operons. INTRODUCTION Alkaline phosphatase and DNAase are two extracellular enzymes whose production is associated with stage I1 of sporulation in Bacillus subtilis. Mutants blocked early in stage I1 produce neither of the enzymes; those blocked later produce both (Waites et al., 1970; Piggot & Coote, 1976; Akrigg & Mandelstam, 1978; Young & Mandelstam, 1979). It is not known whether they play a direct role in sporulation or whether they belong to a category of secondary events (Coote & Mandelstam, 1973). The question could be settled by isolating mutants in which the structural genes for the enzymes are damaged, but, in spite of considerable effort, no such mutants have been found (Grant, 1974; Porter & Mandelstam, 1982). The specific associations of alkaline phosphatase and DNAase with stage I1 could be explained in one of two ways. First, the genes for the enzymes might be included in operons that lie in the main sequence whose successive expression is necessary for the formation of the spore. Alternatively, they might lie on operons unconnected with the main sequence. These might be switched on either by the same regulator molecule that activates an operon in the main sequence or by some other molecule such as an intermediary metabolite generated by some biochemical change, e.g. enzymic action of a protein coded for in the main sequence. This paper describes the phenotypic consequences of a number of closely linked mutations in the spoIIA locus. Earlier work had shown that mutations in this locus result not only in asporogeny or oligosporogeny but also in failure to produce alkaline phosphatase (Coote, 1972a; Piggot, 1973). It was subsequently shown that there is also a failure to produce extracellular DNAase (Akrigg & Mandelstam, 1978). None of the later sporulation events, e.g. production of glucose dehydrogenase or dipicolinic acid, occurred in these mutants. These results all suggest that spoIIA is a ‘main-stream’ locus. More recently, Yudkin & Turley (1981) showed that mutations in one part of the locus could be partly suppressed by another mutation elsewhere in the same locus and that this altered not only the degree of oligosporogeny but the rate at which sporulation occurred. Furthermore, Liu et al. (1982) cloned part of the spoIIA locus on a plasmid and demonstrated that this plasmid would complement mutations that had been shown by Yudkin & Turley (1981) to lie at one end of the locus, whereas mutations lying at the other end could not be complemented. It thus appears that the spoIIA locus contains at least two separate genes. The experiments described in this paper show that closely linked mutations within either of the putative genes can produce quite different effects on alkaline phosphatase, DNAase and sporulation. They also affect in different ways the stability of the cells in sporulation medium. 0022-1287/83/0001-0990 $02.00 0 1983 SGM