SummaryRapid proteolysis plays an important role in regulation of gene expression. Proteolysis of the phage CII transcriptional activator plays a key role in the lysis-lysogeny decision by phage . Here we demonstrate that the E. coli ATP-dependent protease FtsH, the product of the host ftsH/hflB gene, is responsible for the rapid proteolysis of the CII protein. FtsH was found previously to degrade the heat-shock transcription factor 32 . Proteolysis of 32 requires, in vivo, the presence of the DnaK-DnaJ-GrpE chaperone machine. Neither DnaK-DnaJ-GrpE nor GroEL-GroES chaperone machines are required for proteolysis of CII in vivo. Purified FtsH carries out specific ATP-dependent proteolysis of CII in vitro. The degradation of CII is at least 10-fold faster than that of 32 . Electron microscopy revealed that purified FtsH forms ringshaped structures with a diameter of 6-7 nm.
ATP-dependent proteases, like FtsH (HflB), recognize specific protein substrates. One of these is the CII protein, which plays a key role in the phage lysis-lysogeny decision. Here we provide evidence that the conserved C-terminal end of CII acts as a necessary and sufficient cis-acting target for rapid proteolysis. Deletions of this conserved tag, or a mutation that confers two aspartic residues at its C terminus do not affect the structure or activity of CII. However, the mutations abrogate CII degradation by FtsH. We have established an in vitro assay for the CIII protein and demonstrated that CIII directly inhibits proteolysis by FtsH to protect CII and CII mutants from degradation. Phage carrying mutations in the C terminus of CII show increased frequency of lysogenization, which indicates that this segment of CII may itself be sensitive to regulation that affects the lysis-lysogeny development. In addition, the region coding for the C-terminal end of CII overlaps with a gene that encodes a small antisense RNA called OOP. We show that deletion of the end of the cII gene can prevent OOP RNA, supplied in trans, interfering with CII activity. These findings provide an example of a gene that carries a region that modulates stability at the level of mRNA and protein. Proteolysis by ATP-dependent proteases is an important mechanism for the rapid control of gene activity, the removal of unfolded inactive proteins, and the elimination of incomplete polypeptides. In bacteria, proteolysis acts on key regulatory transcription factors affecting the heat shock ( 32 ), stationary phase ( s ), and the SOS DNA repair system (LexA) responses, capsular polysaccharide biosynthesis, and the control of the lysis-lysogeny decision of phage (1). FtsH is a membranebound ATP-dependent protease in which the ATPase domain and the protease domain are linked in one polypeptide (2). FtsH orthologs are found within mitochondria and chloroplasts in higher organisms (3, 4). The number of native proteins known to be substrates for FtsH is rather small and includes the heat shock sigma factor 32 , SecY, YccA, subunit a of the membraneembedded F 0 part of the H ϩ -ATPase, as well as phage CII, CIII, and Xis proteins (see ref. 5). However, the signal(s) by which these native substrates are recognized by FtsH is not known. FtsH, as well as Tsp, ClpAP, and ClpXP recognize the SsrA peptide tag that is added to the C terminus of incomplete proteins by trans-translation (6).The lysogenic response established after infection of Escherichia coli by the temperate bacteriophage requires the initial synthesis of the CI repressor from the pE promoter and the integration protein Int, from the pI promoter. In addition, establishment of viable lysogenic cells requires the expression of the paQ promoter that inhibits lytic gene expression. The phage CII protein, which is 97 amino acid residues long, plays a key initiating role in these processes by activating the pE, pI, and paQ promoters (7). CII itself is regulated at many levels: transcription, translatio...
The BENr gene of Candida albicans, which confers resistance on susceptible strains of Saccharomyces cerevisiae to six structurally and functionally unrelated drugs, was described recently (R. Ben-Yaacov, S. Knoller, G. Caldwell, J. M. Becker, and Y. Koltin, Antimicrob. Agents Chemother. 38:648-652, 1994). This gene bears similarity to membrane proteins encoding antibiotic resistance in prokaryotes and eukaryotes. The effect of disruption of this gene on viability and drug susceptibility was determined. The results indicate that the gene is not essential but its inactivation leads to susceptibility to three of the four drugs tested. Inactivation of this gene did not increase the susceptibility of the mutant to benomyl, suggesting that C. albicans has other mechanisms of resistance, some of which may be additional efflux pumps that confer resistance to this tubulin-destabilizing agent.
We isolated and characterized a new Escherichia coli gene, htpX. The htpX gene has been localized at min 40.3 on the chromosome. We determined its transcription and translation start site. htpX expresses a 32-kDa protein from a monocistronic transcript; expression of this protein is induced by temperature upshift. htpX is expressed from a or32-dependent promoter and is thus part of the heat shock regulon. Cells carrying a htpX gene disruption grow well at all temperatures and under all conditions tested and have no apparent phenotype. However, cells which overexpress a truncated form of the protein display a higher rate of degradation of puromycyl peptides.The heat shock response consists in a transient increase, after temperature upshift, in the synthesis of a small subset of proteins, the heat shock proteins (hsps). This response has been observed in all cells so far examined (reviewed in reference 31). In Escherichia coli, temperature upshift, as well as other treatments such as UV irradiation, bacteriophage infection, and overproduction of abnormal proteins, leads to the preferential synthesis of at least 17 hsps (reviewed in reference 38). The central regulator of the heat shock response in E. coli is the heat shock gene-specific RNA polymerase subunit, cr32 (22, 23).Some hsps have been highly conserved throughout evolution. Three E. coli hsps, GroEL, DnaK, and C62.5, are homologous to the mitochondrial HSP58 protein and to the eukaryotic HSP70 and HSP90 protein families, respectively (5, 6, 35). In contrast, the small hsps, although often conserved within the same organism, display little similarity between different organisms (32).GroE is involved in the assembly of multiprotein complexes (15,20), and DnaK, DnaJ, and GrpE have been shown to participate in the refolding of denatured proteins (14). In addition, strains carrying mutations in these genes are defective in the proteolysis of abnormal proteins (48). The Lon protease, which degrades abnormal proteins in the cell (21, 34), is a heat shock protein as well (16). A common denominator of most conditions that induce the synthesis of the hsps is thought to be the appearance of unfolded proteins in the cell (4,17,41). It is possible that the primary role of the heat shock response is to remove unfolded proteins from the cell, either by refolding or by degrading them.The lysogenization process of bacteriophage A involves several phage proteins, such as the CII transcriptional activator, which is required for the initiation of transcription of the repressor and integrase genes (45), and the CIII protein, whose role is to stabilize CII (3, 24). Several host genes, e.g., rnc (2) and hfl (7), are involved in this process as well. We were interested in isolating additional host genes participating in the regulation of phage lysogenization.A screening procedure aimed at isolating such genes from
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