Table of Contents Supplementary Methods General remarks Synthesis of RcPNA Cytotoxicity against mammalian cell lines Supplementary Figures and Tables Supplementary Table 1: Details on mass spectrometric identification of protein excised from preparative 2D gels. SupplementaryTable 2: Marker proteins induced after FcPNA and RcPNA treatment. Induction factors are displayed as averages over three independently performed biological experiments.

Proteolysis of regulatory proteins or key enzymes of biosynthetic pathways is a universal mechanism to rapidly adjust the cellular proteome to particular environmental needs. Among the five energy-dependent AAA(+) proteases in Escherichia coli, FtsH is the only essential protease. Moreover, FtsH is unique owing to its anchoring to the inner membrane. This review describes the structural and functional properties of FtsH. With regard to its role in cellular quality control and regulatory circuits, cytoplasmic and membrane substrates of the FtsH protease are depicted and mechanisms of FtsH-dependent proteolysis are discussed.

Escherichia coli RidA is a member of a structurally conserved, yet functionally highly diverse protein family involved in translation inhibition (human), Hsp90-like chaperone activity (fruit fly) and enamine/imine deamination (Salmonella enterica). Here, we show that E. coli RidA modified with HOCl acts as a highly effective chaperone. Although activation of RidA is reversed by treatment with DTT, ascorbic acid, the thioredoxin system and glutathione, it is independent of cysteine modification. Instead, treatment with HOCl or chloramines decreases the amino group content of RidA by reversibly N-chlorinating positively charged residues. N-chlorination increases hydrophobicity of RidA and promotes binding to a wide spectrum of unfolded cytosolic proteins. Deletion of ridA results in an HOCl-sensitive phenotype. HOCl-mediated N-chlorination thus is a cysteine-independent post-translational modification that reversibly turns RidA into an effective chaperone holdase, which plays a crucial role in the protection of cytosolic proteins during oxidative stress.

SummaryLipopolysaccharide (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 σ σ σ σ 32 (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 FtsHdependent degradation.

Background: Only few substrates of the essential membrane-anchored protease FtsH are known. Results: New cytoplasmic and membrane-bound substrates of FtsH were trapped in vivo. Conclusion: FtsH is involved in the sulfatation of molecules, D-amino acid metabolism, and adaptation to anaerobiosis and stress conditions. Significance: The novel FtsH substrates significantly expand our knowledge on the biological functions of this fundamentally important protease.

The attachment of lipids to C-or N-terminally positioned lysine side-chain amino groups increases the activity of a short synthetic (Arg-Trp) 3 antimicrobial peptide significantly, making these peptides even active against pathogenic Gram-negative bacteria. Thus, a peptide with strong activity against S. aureus (1.1−2 μM) and good activity against A. baumannii and P. aeruginosa (9−18 μM) was identified. The most promising peptide causes 50% hemolysis at 285 μM and shows some selectivity against human cancer cell lines. Interestingly, the increased activity of ferrocenoylated peptides is mostly due to the lipophilicity of the organometallic fragment.

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The outer membrane is the first line of defense for Gram-negative bacteria and serves as a major barrier for antibiotics and other harmful substances. The biosynthesis of lipopolysaccharides (LPS), the essential component of the outer membrane, must be tightly controlled as both too much and too little LPS are toxic. In Escherichia coli, the cellular level of the key enzyme LpxC, which catalyzes the first committed step in LPS biosynthesis, is adjusted by proteolysis carried out by the essential and membrane-bound protease FtsH. Here, we demonstrate that LpxC is degraded in a growth rate-dependent manner with half-lives between 4 min and >2 h. According to the cellular demand for LPS biosynthesis, LpxC is degraded during slow growth but stabilized when cells grow rapidly. Disturbing the balance between LPS and phospholipid biosynthesis in favor of phospholipid production in an E. coli strain encoding a hyperactive FabZ protein abolishes growth rate dependency of LpxC proteolysis. Lack of the alternative sigma factor RpoS or inorganic polyphosphates, which are known to mediate growth rate-dependent gene regulation in E. coli, did not affect proteolysis of LpxC. In contrast, absence of RelA and SpoT, which synthesize the alarmone (p)ppGpp, deregulated LpxC degradation resulting in rapid proteolysis in fast-growing cells and stabilization during slow growth. Our data provide new insights into the essential control of LPS biosynthesis in E. coli.