Structure and function of the Bacillus SpoIIE protein and its localization to sites of sporulation septum assembly Imrich Bara ´k, 1,² Jaideep Behari, 1 Gabriela Olmedo, 2 Plinio Guzma ´n, 2 David P. Brown, 1 Elda Castro, 2 DeEtte Walker, 1 Janet Westpheling 1 and Philip Youngman 1 * 1 Department of Genetics, University of Georgia, Athens, Georgia 30602, USA. 2 Departamento de Ingenierı ´ a Gene ´ tica, CINVESTAV, Unidad Irapuato, GTO, Mexico. Summary Functioning of the spoIIE locus of Bacillus subtilis is required for formation of a normal polar septum dur- ing sporulation and for activation of the transcription factor F , which directs early forespore-specific gene expression. We have determined the DNA sequence of the wild type and several mutant alleles of the spoIIE gene of B. subtilis and sequenced a substantial portion of its presumptive homologue in Bacillus megaterium. We show that the spoIIE locus encodes a single large protein with a predicted molecular mass of 92 kDa. Each of five point-mutation alleles, which have traditionally defined the locus, and two transposon-generated mutations were shown to fall within the coding sequence for the 92 kDa gene pro- duct or within sequences expected to be required for its expression. The amino-terminal portion of the pre- dicted SpoIIE gene product, comprising approxi- mately 40% of the protein, is extremely hydrophobic and is expected to contain up to 12 membrane-span- ning segments. The remainder of the protein contains no hydrophobic segments long enough to span a lipid bilayer and is therefore presumed to comprise one or more globular, aqueous-phase exposed domains. An in-frame fusion joining the 3 end of the B. megaterium spoIIE coding sequence to the 5 end of gfp, a gene encoding the green fluorescent protein (GFP) of Aquorea victoria, resulted in a strong, sporulation- specific fluorescent signal localized to the sites of sporulation septum assembly. We speculate that SpoIIE plays a role in assembling the sporulation sep- tum, perhaps determining the special properties of the structure that permit intercompartment signalling during development. Introduction At stage II in the morphogenetic programme of sporulation in Bacillus species the developing cell is partitioned into compartments of unequal size as the result of an asym- metric septation event (Piggot et al., 1994). Except for the fact that the sporulation septum contains significantly less peptidoglycan, it resembles the symmetrically posi- tioned septum that defines the location of the cell-division cleft during growth by binary fission (Illing and Errington, 1991). At least some of the genes required for synthesis of the cell division septum are also required for synthesis of the sporulation septum (Lutkenhaus, 1994), indicating that both septum types may be fundamentally similar in their structure and mode of assembly. Nevertheless, the sporulation septum clearly has properties which distin- guish it in structural and functional terms from the division septum. The division septum is a participant in cytokinesis, with the peptidoglycan component splitting bilaterally as daughter cells separate from one another. In contrast, the sporulation septum is a participant in the engulfment process, during which most or all of the peptidoglycan that is initially detectable by electron microscopy within the septum is removed (Holt et al., 1975). Asymmetric septation at stage II coincides with, and may actively initiate, the establishment of separate pro- grammes of gene expression in the two sporangium com- partments (Losick and Stragier, 1992). The forespore- specific programme is apparently initiated by the activation of F , a sigma factor which is present in both compart- ments but which is held inactive in the mother cell through an association with the anti-sigma factor SpoIIAB (Schmidt et al., 1990). In the forespore, SpoIIAB is prevented from inhibiting F by another protein, SpoIIAA, which is an alter- native binding partner for SpoIIAB (Duncan and Losick, 1993). Results from in vitro studies have led to the hypothesis that choice of binding partners for SpoIIAB is controlled by the ratio of ATP:ADP in the two sporangium compartments (Alper et al., 1994). How a differential between the mother cell and forespore in ATP:ADP ratio might be created is unknown. Although mutations in several genetic loci are known Molecular Microbiology (1996) 19(5), 1047–1060 1996 Blackwell Science Ltd Received 30 July, 1995; revised 10 October, 1995; accepted 13 October, 1995. ²Present address: Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovak Republic. *For correspondence. E-mail youngman@bscr.uga.edu; Tel. (706) 542 1417; Fax (706) 542 1417.