Thomas Wiegert

The Bacillus subtilisσW anti‐sigma factor RsiW is degraded by intramembrane proteolysis through YluC

The Bacillus subtilisσW regulon is induced by different stresses such as alkaline shock, salt shock, phage infection and certain antibiotics that affect cell wall biosynthesis. The activity of the alternative, extracytoplasmic function (ECF) sigma factor σW is modulated by a specific anti‐sigma factor (RsiW or YbbM) encoded by the rsiW (ybbM) gene located immediately downstream of sigW. The RsiW membrane topology was determined, and a specific reporter system for RsiW function was constructed. Experiments using the yeast two‐hybrid system suggested a direct interaction of σW with the cytoplasmic part of RsiW. Analysis of truncated forms of the RsiW protein revealed that σW induction by alkaline shock is dependent on both the transmembrane and the extracytoplasmic domain of RsiW. Western blot and pulse–chase experiments demonstrated degradation of RsiW after an alkaline shock. A B. subtilis mutant strain deleted for the Escherichia coli yaeL orthologue yluC, encoding a transmembrane protease, was defective in inducing a σW‐controlled promoter after alkaline shock and accumulated a membrane‐bound truncated form of RsiW, suggesting that the activity of σW is controlled by the proteolysis of RsiW by at least two different proteolytic steps.

SsrA-Mediated Tagging in Bacillus subtilis

A general mechanism in bacteria to rescue stalled ribosomes involves a stable RNA encoded by the ssrA gene. This RNA, termed tmRNA, encodes a proteolytic peptide tag which is cotranslationally added to truncated polypeptides, thereby targeting them for rapid proteolysis. To study this ssrA-mediated mechanism in Bacillus subtilis, a bipartite detection system was constructed that was composed of the HrcA transcriptional repressor and the bgaB reporter gene coding for a heat-stable beta-galactosidase fused to an HrcA-controlled promoter. After the predicted proteolysis tag was fused to HrcA, the reporter beta-galactosidase was expressed constitutively at a high level due to the instability of the tagged HrcA. Replacement of the two C-terminal alanine residues of the tag by aspartate rendered the repressor stable. Replacement of the hrcA stop codon by a transcriptional terminator sequence rendered the protein unstable; this was caused by trans translational addition of the proteolytic tag. Inactivating the B. subtilis ssrA or smpB (yvaI) gene prevented the trans translational tagging reaction. Various protease-deficient strains of B. subtilis were tested for proteolysis of tagged HrcA. HrcA remained stable only in clpX or clpP knockouts, which suggests that this ATP-dependent protease is primarily responsible for the degradation of SsrA-tagged proteins in B. subtilis.

Development of a New Integration Site within the Bacillus subtilis Chromosome and Construction of Compatible Expression Cassettes

The Bacillus subtilis lacA gene, coding for beta-galactosidase, has been explored as a new site able to accept DNA sequences from nonreplicating delivery vectors. Two such delivery expression vectors have been constructed and shown to be useful in obtaining regulated expression from the chromosomal location. In another experiment, it was shown that the integration of a regulatory gene at the lacA locus was able to control the expression of a transcriptional fusion at the amyE locus. These experiments demonstrate that both integration sites can be used simultaneously to obtain regulated expression of desired genes.

Construction and Application of Epitope- and Green Fluorescent Protein-Tagging Integration Vectors for Bacillus subtilis

ABSTRACT Here we describe the construction and application of six new tagging vectors allowing the fusion of two different types of tagging sequences, epitope and localization tags, to any Bacillus subtilis protein. These vectors are based on the backbone of pMUTIN2 and replace the lacZ gene with tagging sequences. Fusion of the tagging sequences occurs by PCR amplification of the 3′ terminal part of the gene of interest (about 300 bp), insertion into the tagging vector in such a way that a fusion protein will be synthesized upon integration of the whole vector via homologous recombination with the chromosomal gene. Three of these tagging sequences (FLAG, hemagglutinin, and c-Myc) allow the covalent addition of a short epitope tag and thereby detection of the fusion proteins in immunoblots, while three other tags (green fluorescent protein+, yellow fluorescent protein, and cyan fluorescent protein) are helpful in assigning proteins within one of the compartments of the cell. The versatility of these vectors was demonstrated by fusing these tags to the cytoplasmically located HtpG and the inner membrane protein FtsH.

Specificity of signal peptide recognition in Tat-dependent bacterial protein translocation

The bacterial twin arginine translocation (Tat) pathway translocates across the cytoplasmic membrane folded proteins which, in most cases, contain a tightly bound cofactor. Specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif, S/T-R-R-X-F-L-K, direct these proteins to the Tat translocon. The glucose-fructose oxidoreductase (GFOR) of Zymomonas mobilis is a periplasmic enzyme with tightly bound NADP as a cofactor. It is synthesized as a cytoplasmic precursor with an amino-terminal signal peptide that shows all of the characteristics of a typical twin arginine signal peptide. However, GFOR is not exported to the periplasm when expressed in the heterologous host Escherichia coli, and enzymatically active pre-GFOR is found in the cytoplasm. A precise replacement of the pre-GFOR signal peptide by an authentic E. coli Tat signal peptide, which is derived from pre-trimethylamine N-oxide (TMAO) reductase (TorA), allowed export of GFOR, together with its bound cofactor, to the E. coli periplasm. This export was inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not by sodium azide, and was blocked in E. coli tatC and tatAE mutant strains, showing that membrane translocation of the TorA-GFOR fusion protein occurred via the Tat pathway and not via the Sec pathway. Furthermore, tight cofactor binding (and therefore correct folding) was found to be a prerequisite for proper translocation of the fusion protein. These results strongly suggest that Tat signal peptides are not universally recognized by different Tat translocases, implying that the signal peptides of Tat-dependent precursor proteins are optimally adapted only to their cognate export apparatus. Such a situation is in marked contrast to the situation that is known to exist for Sec-dependent protein translocation.

The Substitution of a Single Amino Acid Residue (Ser-116 → Asp) Alters NADP-containing Glucose-Fructose Oxidoreductase ofZymomonas mobilis into a Glucose Dehydrogenase with Dual Coenzyme Specificity

Glucose-fructose oxidoreductase (GFOR, EC1.1.1.99.-) from the Gram-negative bacterium Zymomonas mobilis contains the tightly bound cofactor NADP. Based on the revision of the gfo DNA sequence, the derived GFOR sequence was aligned with enzymes catalyzing reactions with similar substrates. A novel consensus motif (AGKHVXCEKP) for a class of dehydrogenases was detected. From secondary structure analysis the serine-116 residue of GFOR was predicted as part of a Rossmann-type dinucleotide binding fold. An engineered mutant protein (S116D) was purified and shown to have lost tight cofactor binding based on (a) altered tryptophan fluorescence; (b) lack of NADP liberation through perchloric acid treatment of the protein; and (c) lack of GFOR enzyme activity. The S116D mutant showed glucose dehydrogenase activity (3.6 ± 0.1 units/mg of protein) with both NADP and NAD as coenzymes (K m for NADP, 153 ± 9 μm; for NAD, 375 ± 32 μm). The single site mutation therefore altered GFOR, which in the wild-type situation contains NADP as nondissociable redox cofactor reacting in a ping-pong type mechanism, to a dehydrogenase with dissociable NAD(P) as cosubstrate and a sequential reaction type. After prolonged preincubation of the S116D mutant protein with excess NADP (but not NAD), GFOR activity could be restored to 70 units/mg, one-third of wild-type activity, whereas glucose dehydrogenase activity decreased sharply. A second site mutant (S116D/K121A/K123Q/I124K) showed no GFOR activity even after preincubation with NADP, but it retained glucose dehydrogenase activity (4.2 ± 0.2 units/mg of protein).

The Absence of FtsH Metalloprotease Activity Causes Overexpression of the σW-Controlled pbpE Gene, Resulting in Filamentous Growth of Bacillus subtilis

FtsH is a membrane-bound and energy-dependent metalloprotease in bacteria which is involved in the posttranslational control of the activity of a variety of important transcription factors and in the degradation of uncomplexed integral membrane proteins. For Bacillus subtilis, little is known about the target proteins of FtsH protease. Its gene is not essential, but knockout strains display a pleiotropic phenotype including sensitivity toward salt and heat stress, defects in sporulation and competence, and largely filamentous growth. Comparison of the intracellular proteomes of wild-type and ftsH knockout strains revealed that at least nine proteins accumulated in the absence of ftsH, four of which could be identified. Two of these proteins turned out to be members of the sigma(W) regulon. Accumulation of one of these sigma(W)-controlled proteins, the penicillin-binding protein PBP4*, was analyzed in more detail. We could show that PBP4* is not a proteolytic substrate of FtsH and that its overproduction is due to the enhanced transcription of its gene (pbpE) in ftsH null mutants. The filamentous growth phenotype of DeltaftsH strains was abolished in a DeltaftsH DeltapbpE double knockout. In ftsH wild-type strains with the pbpE gene under regulatable control, pbpE overexpression caused filamentation of the cells. DNA macroarray analysis revealed that most genes of the sigma(W) regulon are transcribed at elevated levels in an ftsH mutant. The influence of FtsH on sigma(W)-controlled genes is discussed.

Export of the periplasmic NADP-containing glucose-fructose oxidoreductase of Zymomonas mobilis

Abstract Glucose-fructose oxidoreductase (GFOR) of the gram-negative bacterium Zymomonas mobilis is a periplasmic enzyme with the tightly bound cofactor NADP. The preprotein carries an unusually long N-terminal signal sequence of 52 amino acid residues. A sorbitol-negative mutant strain (ACM3963) was found to be deficient in GFOR activity and was used for the expression of plasmid-borne copies of the wild-type gfo gene or of alleles encoding alterations in the signal sequence of the pre-GFOR protein. Z. mobilis cells with the wild-type gfo allele translocated pre-GFOR, at least partially, via the Sec pathway since CCCP (carboxylcyanide-m-chlorophenylhydrazone; uncoupler of proton motive force) or sodium azide (inhibitor of SecA) abolished the processing of GFOR. A gfo allele with the hydrophobic region of the signal sequence removed (residues 32–46; Δ32–46) led to a protein that was no longer processed, but showed full enzymatic activity (180 U/mg) and had the cofactor NADP firmly bound. A deletion in the n-region of the signal sequence (residues 2–20; Δ2–20) or exchange of the entire GFOR signal sequence with the signal sequence of gluconolactonase of Z. mobilis led to active and processed GFOR. Strain ACM3963 could not grow in the presence of high sugar concentrations (1 M sucrose) unless sorbitol was added. The presence of the plasmid-borne gfo wild-type allele or of the Δ2–20 deletion led to the restoration of growth on media with 1 M sucrose, whereas the presence of the Δ32–46 deletion led to a growth behavior similar to that of strain ACM3963, with no sorbitol formation from sucrose.

Regulation of the Bacillus subtilis Heat Shock Gene htpG Is under Positive Control

The heat shock genes of Bacillus subtilis are assigned to four classes on the basis of their regulation mechanisms. While classes I and III are negatively controlled by two different transcriptional repressors, class II is regulated by the alternative sigma factor sigma(B). All heat shock genes with unidentified regulatory mechanisms, among them htpG, constitute class IV. Here, we show that expression of htpG is under positive control. We identified a DNA sequence (GAAAAGG) located downstream of the sigma(A)-dependent promoter of htpG. The heat inducibility of the promoter could be destroyed by inversion, nucleotide replacements, or removal of this DNA sequence. Fusion of this sequence to the vegetative lepA promoter conferred heat inducibility. Furthermore, we were able to show that the heat induction factor is dependent on the absolute temperature rather than the temperature increment and that nonnative proteins within the cytoplasm fail to induce htpG.