SummaryThe suppressor mutation, named sfhC21, that allows Escherichia coli ftsH null mutant cells to survive was found to be an allele of fabZ encoding R-3-hydroxyacyl-ACP dehydrase, involved in a key step of fatty acid biosynthesis, and appears to upregulate the dehydrase. The ftsH1(Ts) mutation increased the amount of lipopolysaccharide at 42ЊC. This was accompanied by a dramatic increase in the amount of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase [the lpxC (envA) gene product] involved in the committed step of lipid A biosynthesis. Pulse-chase experiments and in vitro assays with purified components showed that FtsH, the AAA-type membrane-bound metalloprotease, degrades the deacetylase. Genetic evidence also indicated that the FtsH protease activity for the deacetylase might be affected when acyl-ACP pools were altered. The biosynthesis of phospholipids and the lipid A moiety of lipopolysaccharide, both of which derive their fatty acyl chains from the same R-3-hydroxyacyl-ACP pool, is regulated by FtsH.

Escherichia coliFtsH is an ATP-dependent protease that belongs to the AAA protein family. The second region of homology (SRH) is a highly conserved motif among AAA family members and distinguishes these proteins in part from the wider family of Walker-type ATPases. Despite its conservation across the AAA family of proteins, very little is known concerning the function of the SRH. To address this question, we introduced point mutations systematically into the SRH of FtsH and studied the activities of the mutant proteins. Highly conserved amino acid residues within the SRH were found to be critical for the function of FtsH, with mutations at these positions leading to decreased or abolished ATPase activity. The effects of the mutations on the protease activity of FtsH correlated strikingly with their effects on the ATPase activity. The ATPase-deficient SRH mutants underwent an ATP-induced conformational change similar to wild type FtsH, suggesting an important role for the SRH in ATP hydrolysis but not ATP binding. Analysis of the data in the light of the crystal structure of the hexamerization domain of Nethylmaleimide-sensitive fusion protein suggests a plausible mechanism of ATP hydrolysis by the AAA ATPases, which invokes an intermolecular catalytic role for the SRH.

DNA-damage-induced SOS mutations arise when Escherichia coli DNA polymerase (pol) V, activated by a RecA nucleoprotein filament (RecA*), catalyses translesion DNA synthesis. Here we address two longstanding enigmatic aspects of SOS mutagenesis, the molecular composition of mutagenically active pol V and the role of RecA*. We show that RecA* transfers a single RecA-ATP stoichiometrically from its DNA 3′-end to free pol V (UmuD′ 2 C) to form an active mutasome (pol V Mut) with the composition UmuD′ 2 C-RecA-ATP. Pol V Mut catalyses TLS in the absence of RecA* and deactivates rapidly upon dissociation from DNA. Deactivation occurs more slowly in the absence of DNA synthesis, while retaining RecA-ATP in the complex. Reactivation of pol V Mut is triggered by replacement of RecA-ATP from RecA*. Thus, the principal role of RecA* in SOS mutagenesis is to transfer RecA-ATP to pol V, and thus generate active mutasomal complex for translesion synthesis.Pol V is a low-fidelity DNA polymerase 1,2 induced as part of the SOS regulon in E. coli in response to DNA damage 3 . The replicative polymerase, pol III, typically stalls when it encounters a DNA template lesion, arresting movement of the replication fork. One pathway that enables restoration of fork movement involves pol V, which replaces pol III on the sliding β-clamp 4-6 and catalyses translesion DNA synthesis (TLS). Pol V copies numerous types of lesions 7 , but in a mutagenic manner 8,9 . After TLS, pol III resumes normal replication.The pol V complex consists of UmuD′ 2 C 10,11 . Both in vitro 1,2,12,13 and in vivo [14][15][16] , pol V activity requires the assembly of an active RecA filament on single-stranded (ss) DNA, termed RecA* 17 . The biological functions of RecA* in strand exchange during homologous recombination and in mediating cleavage of the repressor protein LexA and UmuD during the SOS response are well understood 17 . In contrast, the biochemical role of RecA* in pol-Vdependent mutagenic TLS remains poorly characterized.Proposals for the role of RecA* in TLS have evolved from positioning UmuD′ 2 C on primer/ template (p/t) DNA proximal to a lesion [18][19][20] Formation of activated pol V MutIn earlier studies, either free RecA protein or RecA filament was added to pol V and p/t DNA substrate. Here, we first form RecA* by incubating RecA with biotinylated ssDNA bound to a streptavidin-agarose resin matrix in the presence of ATPγS (adenosine 5′ [γ-thio] triphosphate; see Methods). Pol V (UmuD′ 2 C) is then incubated with RecA* in the absence of p/t DNA, forming an activated pol V species that can be isolated, pol V Mut ( Supplementary Figs 1 and 2). Only once RecA* is removed by centrifugation is p/t DNA added, allowing DNA synthesis in the absence of RecA* (Fig. 1a, b). In these studies, the p/t template is a hairpin with a 3-nucleotide overhang 21 instead of, for example, an oligonucleotide annealed to a ssDNA circle; this prevents formation of activated RecA* on the p/t DNA itself because the hairpin lacks free ssDNA on which RecA* can a...

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SummaryThe heat shock response of Escherichia coli is regulated by the cellular level and the activity of 32 , an alternative sigma factor for heat shock promoters. FtsH, a membrane-bound AAA-type metalloprotease, degrades 32 and has a central role in the control of the 32 level. The ftsH null mutant was isolated, and establishment of the ⌬ftsH mutant allowed us to investigate control mechanisms of the stability and the activity of 32 separately in vivo. Loss of the FtsH function caused marked stabilization and consequent accumulation of 32 (Ϸ20-fold of the wild type), leading to the impaired downregulation of the level of 32 . Surprisingly, however, ⌬ftsH cells express heat shock proteins only two-to threefold higher than wild-type cells, and they also show almost normal heat shock response upon temperature upshift. These results indicate the presence of a control mechanism that downregulates the activity of 32 when it is accumulated. Overproduction of DnaK /J reduces the activity of 32 in ⌬ftsH cells without any detectable changes in the level of 32 , indicating that the DnaK chaperone system is responsible for the activity control of 32 in vivo. In addition, CbpA, an analogue of DnaJ, was demonstrated to have overlapping functions with DnaJ in both the activity and the stability control of 32 .