Molecular Microbiology (1991) 5(1), 173-185 0960382X9100021L The oligopeptide transport system of Bacillus subtilis plays a role in the initiation of sporulation M. Perego,^^ C. F. Higgins,^ S. R. Pearce,^ M. P. Gallagher2§ and J. A. Hoch^* 'Division of Cellular Biology, Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, California 92037, USA. ^Imperial Cancer Research Fund Laboratories. University of Oxford, Institute of Molecular Medicine, John Radcliffe Hospital. Oxford 0X3 9DU, UK. Summary Bacillus subtilis spoOK mutants are blocked at the first step in sporulatJon. The spoOK strain was found to contain two mutations: one was linked to the trpS locus, and the other was elsewhere on the chromo- some. The mutation linked to frpS was responsible for the sporulation defect (spo ). The unlinked mutation enhanced this sporulation deficiency but had no phe- notype on its own. The spo mutation was located in an operon of five genes highly homologous to the oli- gopeptide transport (Opp) system of Gram-negative species. Studies with toxic peptide analogues showed that this operon does indeed encode a peptide-tran- sport system. However, unlike the Opp system of Salmonella typhimurium, one of the two ATP-binding proteins, OppF, was not required for peptide transport or for sporulation. The OppA peptide-binding protein, which is periplasmically located in Gram-negative species, has a signal sequence characteristic of lipo- proteins with an amino-terminal lipo-amino acid anchor. Cellular location studies revealed that OppA was associated with the cell during exponential growth, but was released into the medium in station- ary phase. A major role of the Opp system in Gram- negative bacteria is the recycling of cell-wall peptides as they are released from the growing peptidogtycan. We postulate that the accumulation of such peptides may play a signaling role tn the initiation of sporu- lation, and that the sporulation defect in opp mutants results from an inability to transport these peptides. Received 11 September, 1990; revised 12 October, 1990, Present addresses: tDipartimento di Genetics e Microbioiogia, Secione di Gene- tics. Unlversita Degli Studi di Pavia. via S. Epitanio, 27100 Pavia, Italy; JDepartment ol Biochemistry. University of Dundee, Dundee DD1 4HN. UK; §Department of Microbiology, University of Edinburgh. King's Build- ings, West Mains Road, Edinburgh EH9 3JG, UK. 'For correspondence, Tel, (619)554 8011; Fax (619) 554 6927, Introduction The initiation of sporulation in Bacillus subtilis requires the functions of severai genes, most of which (when mutated) give a stage zero (SpoO) phenotype (Hoch, 1976). Many of these genes have now been cioned and sequenced and the function of their gene products is beginning to be understood. The spoOH gene codes for a sigma factor required for the transcription of early sporulation genes (Dubnau et ai, 1988). The spoOA and spoOF gene pro- ducts are both response proteins of two-component regulatory systems (Ferrari etal., 1985; Trach etai. 1985) which are subject to activation by phosphorytation (Per- ego eta/., 1989). The spoOB gene is cotranscribed with a gene for an essential GTP-binding protein and may have a role in signal transduction (Trach and Hoch, 1989). The spoOF gene has also been cloned and sequenced and its product identified (Perego and Hoch, 1987). Of the spoO loci identified by classical genetic means (Piggot and Coote, 1976), only the spoOJ and spoOK loci have not been extensively characterized. The spoOK locus was originally defined by a single mutant whose defect was located between the argC4 and metA3 markers on the chromosome (Coote, 1972b). The spoOK" mutant was oligosporogenous and divided to give short square cells (Coote, 1972a). In this communication we show that the original spoOK mutant contains two mutations, one of which enhances the phenotype but is unlinked to the spoOK locus. The spoOK mutation respon- sible for the sporulation-defective phenotype was shown to reside in an operon very similar to the oligopeptide transport operon of Salmonella typhimurium (Hiles ef ai. 1987a). The operon from B. subtilis has been completely sequenced and shown to be involved in peptide uptake. The implications of these findings for the control of sporulation are discussed. Results Linkage analysis trpS-spoOK The spoOK" mutation, spoOK141, was genetically located between the metC and argC loci (Coote, 1972a,b). A second sporulation-defective mutation, spo-834, also mapped to the same chromosomal region (J. A, Hoch, unpublished data). Both mutations were found to cotrans- form with the temperature-sensitive trpS1 marker, with