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Control of cellular processes by regulated proteolysis is conserved among all organisms. FtsH, the only membrane-anchored AAA protease in bacteria, fulfills a variety of regulatory functions. This review focuses on soluble FtsH substrates in Escherichia coli and in other bacteria and outlines emerging substrate recognition principles.

Transcription of most heat shock genes in Escherichia coli is initiated by the alternative sigma factor sigma(32) (RpoH). At physiological temperatures, RpoH is rapidly degraded by chaperone-mediated FtsH-dependent proteolysis. Several RpoH residues critical for degradation are located in the highly conserved region 2.1. However, additional residues were predicted to be involved in this process. We introduced mutations in region C of RpoH and found that a double mutation (A131E, K134V) significantly stabilized RpoH against degradation by the FtsH protease. Single-point mutations at these positions only showed a slight effect on RpoH stability. Both double and single amino acid substitutions did not impair sigma factor activity as demonstrated by a groE-lacZ reporter gene fusion, Western blot analysis of heat shock gene expression and increased heat tolerance in the presence of these proteins. Combined mutations in regions 2.1 and C further stabilized RpoH. We also demonstrate that an RpoH fragment composed of residues 37-147 (including regions 2.1 and C) is degraded in an FtsH-dependent manner. We conclude that in addition to the previously described turnover element in region 2.1, a previously postulated second region important for proteolysis of RpoH by FtsH lies in region C of the sigma factor.

Lipopolysaccharide (LPS) biosynthesis is essential in Gram negative bacteria. LpxC, the key enzyme in LPS formation, catalyses the limiting reaction and controls the ratio between LPS and phospholipids. As overproduction of LPS is toxic, the cellular amount of LpxC must be regulated carefully. The membrane-bound protease FtsH controls the level of LpxC via proteolysis making FtsH the only essential protease of Escherichia coli. We found that the chaperones DnaK and DnaJ co-purified with LpxC. However, degradation of LpxC was DnaK/J-independent in contrast to turnover of the heat shock sigma factor sigma32 (RpoH). The stability of LpxC in a bacterial one-hybrid system suggested that a terminus of LpxC might be important for degradation. Different LpxC truncations and extensions were constructed. Removal of at least five amino acids from the C-terminus abolished degradation by FtsH in vivo. While addition of two aspartic acids to LpxC did not alter its half-life, the exchange of the last two residues against aspartic acids resulted in stabilization. All stable LpxC enzymes were active in vivo as assayed by their high toxicity. Our data demonstrate that the C-terminus of LpxC contains a signal sequence necessary for FtsH-dependent degradation.

Transcription of most heat shock genes in Escherichia coli is initiated by the alternative sigma factor sigma(32) (RpoH). At physiological temperatures, RpoH is rapidly degraded by chaperone-mediated FtsH-dependent proteolysis. Several RpoH residues critical for degradation are located in the highly conserved region 2.1. However, additional residues were predicted to be involved in this process. We introduced mutations in region C of RpoH and found that a double mutation (A131E, K134V) significantly stabilized RpoH against degradation by the FtsH protease. Single-point mutations at these positions only showed a slight effect on RpoH stability. Both double and single amino acid substitutions did not impair sigma factor activity as demonstrated by a groE-lacZ reporter gene fusion, Western blot analysis of heat shock gene expression and increased heat tolerance in the presence of these proteins. Combined mutations in regions 2.1 and C further stabilized RpoH. We also demonstrate that an RpoH fragment composed of residues 37-147 (including regions 2.1 and C) is degraded in an FtsH-dependent manner. We conclude that in addition to the previously described turnover element in region 2.1, a previously postulated second region important for proteolysis of RpoH by FtsH lies in region C of the sigma factor.

The membrane-anchored FtsH protease is essential in Escherichia coli as it adjusts the cellular amount of LpxC, the key enzyme in lipopolysaccharide (LPS) biosynthesis. Both accumulation and depletion of LpxC are toxic to E. coli. By continuous proteolysis of LpxC, FtsH maintains a low concentration of LpxC and, hence, the proper equilibrium between LPS and phospholipids. The C terminus of LpxC is required for turnover. By adding this tail to glutathione-S-transferase (GST) we show that it is necessary but not sufficient for FtsH-mediated degradation. A detailed mutational analysis revealed six non-polar residues in the C terminus of LpxC that are critical for degradation. Alteration of the C-terminal AVLA motif towards the SsrA-like sequence ALAA directed LpxC to other cellular proteases reinforcing the importance of the C-terminal tail for targeting to FtsH. Short C-terminal truncations stabilized LpxC. Most mutations in the C terminus of LpxC left its enzymatic activity intact as was shown by growth assays, microscopy and 2-keto-3-deoxyoctonate (KDO) determination. The critical length of the turnover element was defined by internal deletions. A C-terminal tail of about 20 amino acids length is required for proteolysis of LpxC by FtsH.

Rhodobacter capsulatus was shown to grow efficiently with taurine as sole source of sulfur. We identified a gene region exhibiting similarity to the Escherichia coli tauABC genes coding for a taurine-specific ABC transporter. The R. capsulatus tauABC genes were flanked by two putative operons (orf459-484-590 and cysE-srpI-nifS2) both reading in opposite direction relative to tauABC. Orf459 shows strong similarity to taurine:pyruvate aminotransferase (Tpa) from Bilophila wadsworthia catalyzing the initial transamination during anaerobic taurine degradation, and Orf590 exhibits clear similarity to sulfoacetaldehyde sulfo-lyase from Desulfonispora thiosulfatigenes probably catalyzing the step following the taurine:pyruvate aminotransferase (Tpa) reaction, whereas nifS2 might code for a putative cysteine desulfurase. Expression of R. capsulatus tauABC and nifS2 was inhibited by sulfate, suggesting that tauABC and nifS2 might belong to the same regulon. In contrast, transcription of orf459 was not inhibited by sulfate but was induced by taurine. A tauAB deletion mutant showed significantly reduced growth compared to the wild-type with taurine as sole sulfur source in the presence of serine as a nitrogen source, whereas normal growth was observed in the presence of taurine and ammonium. Deletion of orf459-484-590 completely abolished growth with taurine/serine. Single mutations in any of the three genes resulted in the same phenotype, indicating that all three genes of this putative operon are essential for taurine sulfur utilization in the presence of serine. A model for anaerobic taurine sulfur assimilation in R. capsulatus is discussed.