DegU-P Represses Expression of the Motilityfla-cheOperon inBacillus subtilis

Abstract

Bacillus subtilis implements several adaptive strategies to cope with nutrient limitation experienced at the end of exponential growth. The DegS-DegU two-component system is part of the network involved in the regulation of postexponential responses, such as competence development, the production of exoenzymes, and motility. The degU32(Hy) mutation extends the half-life of the phosphorylated form of DegU (DegU-P); this in turn increases the production of alkaline protease, levan-sucrase, and other exoenzymes and inhibits motility and the production of flagella. The expression of the flagellum-specific sigma factor SigD, of the flagellin gene hag, and of the fla-che operon is strongly reduced in a degU32(Hy) genetic background. To investigate the mechanism of action of DegU-P on motility, we isolated mutants of degU32(Hy) that completely suppressed the motility deficiency. The mutations were genetically mapped and characterized by PCR and sequencing. Most of the mutations were found to delete a transcriptional termination signal upstream of the main flagellar operon, fla-che, thus allowing transcriptional readthrough from the cod operon. Two additional mutations improved the A -dependent promoter sequence of the fla-che operon. Using an electrophoretic mobility shift assay, we have demonstrated that purified DegU binds specifically to the P A promoter region of the fla-che operon. The data suggest that DegU represses transcription of the fla-che operon, and they indicate a central role of the operon in regulating the synthesis and assembly of flagella.Swimming motility in bacteria depends upon the presence on the cell surface of the flagellar organelle, composed of the basal body, the hook, and the filament. The production of flagella is of such adaptive value that most bacterial species are endowed with flagella, despite the high energy requirement for the synthesis of the numerous flagellin monomers that are necessary to build and maintain the flagellar filament.In enterobacteria, the genes involved in flagellar formation are organized into regulons which are arranged into three hierarchical classes. The first class is constituted by the flhDC master operon, whose expression is necessary to turn on class II genes, coding for components of the export machinery and for the hook and basal body. The class II gene fliA encodes 28 , the transcription factor for the class III genes, which include flagellar filament structural genes and the chemotaxis signal transduction system (7,19). In addition, many global regulators, such as CAP, H-NS, H-HU, Lrp, etc., have been reported to affect flagellar synthesis and assembly (5, 13, 24, 34).In Bacillus subtilis, a bona fide master operon is missing and all genes corresponding to the enteric class II are clustered in a single fla-che operon. The expression of the operon depends upon a A -recognized promoter (fla-che P A ), with an additional D -dependent promoter (P D-3 ) playing a minor role (1, 9). Deletion of the fla-che P A promoter renders the cells completely nonmotile, ...

A pivotal role for the response regulator DegU in controlling multicellular behaviour

“…The former detects the signal or stress, whereas the latter controls cellular response, predominantly through gene transcription (Mascher et al, 2006). DegU plays a key role in regulating post-exponential-phase processes in B. subtilis, including activation and inhibition of genetic competence (Dubnau et al, 1994;Kunst et al, 1994;Ogura & Tanaka, 1996), activation and inhibition of motility (Amati et al, 2004;Kobayashi, 2007;Verhamme et al, 2007), activation of degradative enzyme production (Dahl et al, 1992;Msadek et al, 1990), activation of poly-c-glutamic acid production (Stanley & Lazazzera, 2005) and also the activation and inhibition of biofilm formation (Kobayashi, 2007;Verhamme et al, 2007). DegU has regulatory activity in both its unphosphorylated and phosphorylated states.…”

SwrAA activates poly-γ-glutamate synthesis in addition to swarming in Bacillus subtilis

“…Total RNA was isolated from cells grown in LB at 37 uC to OD 600 1.2 as previously described (Amati et al, 2004). RNA template (500 ng) was reverse transcribed using random primers and ThermoScript Reverse Transcriptase (Invitrogen), following the manufacturer's instructions.…”

“…To construct strains with degU32(Hy) or degS200(Hy) mutations, the degS-degU region was amplified with primers loxdegS1 and loxdegU1 from PB5213 and PB5246 chromosomal DNA (Amati et al, 2004) as templates. The products were cloned into the XhoI and HindIII sites of plasmid pLoxSpec (a generous gift from E. Ferrari, Genencor, Palo Alto, CA, USA), thus generating pLoxdegS1 U(Hy) or pLoxdegS200-U1.…”

“…A PCR fragment was amplified from PB5319 (Amati et al, 2004) using primers codYF and nRflgBN, digested with EcoRI and NcoI and inserted between the corresponding sites of pMAD. The resulting plasmid pMADdhs was used to transform PB5370 following the procedure described by Arnaud et al (2004).…”

“…To introduce the dhsA6 mutation (Amati et al, 2004) into PB5249 and PB5370, the shuttle vector pMAD (Arnaud et al, 2004) was used. A PCR fragment was amplified from PB5319 (Amati et al, 2004) using primers codYF and nRflgBN, digested with EcoRI and NcoI and inserted between the corresponding sites of pMAD.…”