A xylose-inducible Bacillus subtilis integration vector and its application

Kim, Lana; Mogk, Axel; Schumann, Wolfgang

doi:10.1016/s0378-1119(96)00466-0

Cited by 190 publications

(172 citation statements)

References 19 publications

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“…To examine whether TepA is a cytosolic or membrane-associated protein, the 3Ј end of the tepA gene was extended with 11 codons, specifying the human c-Myc epitope (37). Next, the myc-tagged tepA gene was placed under the control of the xylose-inducible xylA promoter of plasmid pX (38), resulting in plasmid pXTmyc. Unfortunately, for unknown reasons, the Myc-tagged TepA protein (TepA-Myc) could not be detected in Western blotting experiments with B. subtilis cells transformed with pXTmyc (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Signal Peptide Peptidase- and ClpP-like Proteins of Bacillus subtilis Required for Efficient Translocation and Processing of Secretory Proteins

Bolhuis¹,

Matzen

Hyyryläinen

et al. 1999

Journal of Biological Chemistry

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Signal peptides direct the export of secretory proteins from the cytoplasm. After processing by signal peptidase, they are degraded in the membrane and cytoplasm. The resulting fragments can have signaling functions. These observations suggest important roles for signal peptide peptidases. The present studies show that the Gram-positive eubacterium Bacillus subtilis contains two genes for proteins, denoted SppA and TepA, with similarity to the signal peptide peptidase A of Escherichia coli. Notably, TepA also shows similarity to ClpP proteases. SppA of B. subtilis was only required for efficient processing of pre-proteins under conditions of hyper-secretion. In contrast, TepA depletion had a strong effect on pre-protein translocation across the membrane and subsequent processing, not only under conditions of hyper-secretion. Unlike SppA, which is a typical membrane protein, TepA appears to have a cytosolic localization, which is consistent with the observation that TepA is involved in early stages of the secretion process. Our observations demonstrate that SppA and TepA have a role in protein secretion in B. subtilis. Based on their similarity to known proteases, it seems likely that SppA and TepA are specifically required for the degradation of proteins or (signal) peptides that are inhibitory to protein translocation.

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Section: Resultsmentioning

confidence: 99%

Signal Peptide Peptidase- and ClpP-like Proteins of Bacillus subtilis Required for Efficient Translocation and Processing of Secretory Proteins

Bolhuis¹,

Matzen

Hyyryläinen

et al. 1999

Journal of Biological Chemistry

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“…birA was amplified using oligonucleotides 2245 (59-AATAAGGATC-CACATAGAGGAGTGGCTGAAGACAT-39) and 2246 (59-AATCTG-GATCCCAAGCCAAAAAACCTTCCGTTAC-39) and cloned as a BamHI fragment in pX (Kim et al, 1996). The pX : : birA construct was integrated at amyE.…”

Section: Methodsmentioning

confidence: 99%

Characterization of tRNACys processing in a conditional Bacillus subtilis CCase mutant reveals the participation of RNase R in its quality control

et al. 2010

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We generated a conditional CCase mutant of Bacillus subtilis to explore the participation in vivo of the tRNA nucleotidyltransferase (CCA transferase or CCase) in the maturation of the singlecopy tRNA Cys , which lacks an encoded CCA 39 end. We observed that shorter tRNA Cys species, presumably lacking CCA, only accumulated when the inducible Pspac : cca was introduced into an rnr mutant strain, but not in combination with pnp. We sequenced the tRNA 39 ends produced in the various mutant tRNA Cys species to detect maturation and decay intermediates and observed that decay of the tRNA Cys occurs through the addition of poly(A) or heteropolymeric tails. A few clones corresponding to full-size tRNAs contained either CCA or other C and/or A sequences, suggesting that these are substrates for repair and/or decay. We also observed editing of tRNA Cys at position 21, which seems to occur preferentially in mature tRNAs. Altogether, our results provide in vivo evidence for the participation of the B. subtilis cca gene product in the maturation of tRNAs lacking CCA. We also suggest that RNase R exoRNase in B. subtilis participates in the quality control of tRNA.

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“…Chromosomal DNA of transformants (1012 ecsA : : spec) resistant to spectinomycin was checked by PCR and by Southern blotting using a DIG-labelled DNA probe for ecsA according to standard procedures (Sambrook & Russell, 2005). The ecsA knockout was combined with a transcriptional fusion of the s W -controlled yuaF promoter to lacZ by transforming chromosomal DNA of 1012 ecsA : : spec into B. subtilis JAH12, which is a derivative of BFS233 containing the empty xylose control system of plasmid pX (Kim et al, 1996) integrated in amyQ. As it was not possible to transduce the rasP : : tet construct into B. subtilis strain NCIB3610, a rasP : : spec construct was cloned by replacing the ecsA-59 and ecsA-39 regions of pJH07 with the respective rasP up-and downstream regions that were PCR-amplified using primers (5) 59-GGCCATGTCGACTGTGAT-CGCACAGCTCGGAAC-39, (6) 59-GGCCATGGATCCTATAACTG-TATTCACGAACATACCA-39, (7) 59-GGCCATGAGCTCTTGTCAC-ATGGAACGATATCCAG-39 and (8) 59-GGCCATGAATTCCCTC-ATCGCGAACAAGCGAAG-39, resulting in plasmid pJH08.…”

Section: Methodsmentioning

confidence: 99%

The Bacillus subtilis ABC transporter EcsAB influences intramembrane proteolysis through RasP

et al. 2008

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The Bacillus subtilis s W regulon is induced by different stresses that most probably affect integrity of the cell envelope. The activity of the extracytoplasmic function (ECF) sigma factor s W is modulated by the transmembrane anti-sigma factor RsiW, which undergoes stress-induced degradation in a process known as regulated intramembrane proteolysis, finally resulting in the release of s W and the transcription of s W -controlled genes. Mutations in the ecsA gene, which encodes an ATP binding cassette (ABC) of an ABC transporter of unknown function, block site-2 proteolysis of RsiW by the intramembrane cleaving protease RasP (YluC). In addition, degradation of the cell division protein FtsL, which represents a second RasP substrate, is blocked in an ecsA-negative strain. The defect in s W induction of an ecsA-knockout strain could be partly suppressed by overproducing RasP. A B. subtilis rasP-knockout strain displayed the same pleiotropic phenotype as an ecsA knockout, namely defects in processing a-amylase, in competence development, and in formation of multicellular structures known as biofilms. INTRODUCTIONGenes of Bacillus subtilis controlled by the alternative extracytoplasmic function (ECF) sigma factor s W constitute an antibiosis regulon (Helmann, 2002(Helmann, , 2006 that responds to a variety of envelope stresses such as certain antibiotics (Cao et al., 2002) and antimicrobial peptides (Pietiäinen et al., 2005;Butcher & Helmann, 2006), and also alkaline shock and phage infection . Its activity is modulated by RsiW, a transmembrane anti-sigma factor that sequesters and inactivates s W . Upon a stress signal, RsiW is degraded by the mechanism of regulated intramembrane proteolysis (RIP). In a concerted action, at least three proteases in three different compartments of the cell degrade the RsiW anti-sigma factor in a sequential manner, finally resulting in the release of s W and the transcription of s Wcontrolled genes. The first protease that has been identified to be involved in that process is RasP (regulating anti-sigma factor protease; formerly YluC). RasP belongs to the group of zinc-dependent intramembrane cleaving proteases (iClips) and cleaves RsiW in its transmembrane domain after the extracytoplasmic part of the anti-sigma factor has been removed by a site-1 protease (Schöbel et al., 2004). The site-2 degradation step catalysed by RasP uncovers a cryptic proteolytic tag that ensures further degradation of the RsiW remnant by cytoplasmic proteases, mainly ClpXP (Zellmeier et al., 2006). These two steps are related to RIP of the s E anti-sigma factor RseA of Escherichia coli (Ades, 2004;Alba & Gross, 2004). RasP is an orthologue of E. coli RseP, and the concept of a cryptic proteolytic tag recognized by ClpXP was first described for that system (Akiyama et al., 2004;Flynn et al., 2004). However, there is a marked difference in the site-1 proteolytic step, because no dependency on B. subtilis Deg (Htr) proteases was found and the inducing stress is apparently different. The probable site-1 prot...

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A xylose-inducible Bacillus subtilis integration vector and its application

Cited by 190 publications

References 19 publications

Signal Peptide Peptidase- and ClpP-like Proteins of Bacillus subtilis Required for Efficient Translocation and Processing of Secretory Proteins

Signal Peptide Peptidase- and ClpP-like Proteins of Bacillus subtilis Required for Efficient Translocation and Processing of Secretory Proteins

Characterization of tRNACys processing in a conditional Bacillus subtilis CCase mutant reveals the participation of RNase R in its quality control

The Bacillus subtilis ABC transporter EcsAB influences intramembrane proteolysis through RasP

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