Transcriptional responses during outgrowth of Bacillus subtilis endospores

Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK 1 Author for correspondence: Anne Moir. Tel: +44 114 222 2826. Fax: +44 114 272 8697. e-mail: A.Moir{at}sheffield.ac.uk The Bacillus subtilis 168 genome contains an array of alternative factors, many of which play important roles in reprogramming expression during stress and sporulation. The role of the different factors during outgrowth, when the germinated endospore is converted back to a vegetative cell, is less well characterized. The activity of the alternative factors B , D and H during endospore outgrowth was analysed by Northern blotting and lacZ reporter assays. While D and H were transcriptionally active during outgrowth, B -dependent transcription was not observed until after the first cell division, when growth slowed. Using an IPTG-controllable copy of sigA , an optimal level of expression was required to maintain growth rate at the end of outgrowth. The genes encoding the putative extracytoplasmic function (ECF) factors I , V , W , Z and YlaC were insertionally inactivated using pMUTIN4. These strains, together with sigM and sigX mutants, were tested to determine their role and measure their expression during endospore outgrowth. Transcripts or ß-galactosidase activity were observed for each of the ECF factors early after germination. With the exception of MJH003 ( sigM ), which showed an exacerbated salt stress defect, inactivation of the ECF factor genes did not affect outgrowth in the conditions tested. Keywords: outgrowth, transcription, sigma factor, Bacillus subtilis Abbreviations: ECF, extracytoplasmic function; NB, nutrient broth; SMM, Spizizen minimal medium