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Characterization of the Escherichia coliςE Regulon

2001; Elsevier BV; Volume: 276; Issue: 24 Linguagem: Inglês

10.1074/jbc.m100464200

1083-351X

Claire Dartigalongue, Dominique Missiakas, Satish Raina,

RNA and protein synthesis mechanisms

Escherichia coli responds to the accumulation of misfolded proteins by inducing the transcription of heat shock genes. EςE RNA polymerase controls one of the two heat shock regulons of E. coli. This regulon is activated upon accumulation of misfolded polypeptides in the double membrane envelope of E. coli. ςE (RpoE) is a member of the extracytoplasmic function subfamily of sigma factors. Here we asked how many genes are activated by EςE RNA polymerase and what is the identity of these genes. Using two independent genetic approaches, 20 E. coli promoters were identified which activate reporter gene transcription in a ςE-dependent manner. In all cases examined, a canonical ςE binding site could be revealed upon mapping transcriptional start sites. 10 identified promoters activated the transcription of previously identified genes with four genes acting directly on the folding of E. coli envelope proteins (dsbC, fkpA, skp, andsurA). The remaining promoters transcribed genes that are presumed to encode hitherto unknown extracytoplasmic functions and were named ecf (ecfA–ecfM). Two of these ecf genes were found to be essential for E. coli growth. Escherichia coli responds to the accumulation of misfolded proteins by inducing the transcription of heat shock genes. EςE RNA polymerase controls one of the two heat shock regulons of E. coli. This regulon is activated upon accumulation of misfolded polypeptides in the double membrane envelope of E. coli. ςE (RpoE) is a member of the extracytoplasmic function subfamily of sigma factors. Here we asked how many genes are activated by EςE RNA polymerase and what is the identity of these genes. Using two independent genetic approaches, 20 E. coli promoters were identified which activate reporter gene transcription in a ςE-dependent manner. In all cases examined, a canonical ςE binding site could be revealed upon mapping transcriptional start sites. 10 identified promoters activated the transcription of previously identified genes with four genes acting directly on the folding of E. coli envelope proteins (dsbC, fkpA, skp, andsurA). The remaining promoters transcribed genes that are presumed to encode hitherto unknown extracytoplasmic functions and were named ecf (ecfA–ecfM). Two of these ecf genes were found to be essential for E. coli growth. core RNA polymerase sigma E transcription factor holoenzyme complexed to sigma E regulator of ςE a variant of RseA lacking the first 28 amino acids an allele ofrpoE encoding a mutant of ςE with severely impaired transcriptional activity extracytoplasmic function gene product Heat shock and other environmental stresses result in the misfolding of polypeptides in all cells. Escherichia coliresponds to the accumulation of misfolded polypeptides by activating the transcription of heat shock genes. Heat is a drastic stress that leads to protein unfolding in general and triggers two heat shock responses controlled by two distinct RNA polymerase species in E. coli 1: α2ββ′ς32 and α2ββ′ςE, Eς32, and EςE, respectively (1Gross C.A. Neidhardt F.C. Curtis R.I. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium : Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1996: 1382-1399Google Scholar, 2Missiakas D. Raina S. Georgopoulos C. Lin E.C.C. Lynch S.A. Regulation of Gene Expression in Escherichia coli. R. G. Landes Company, Austin, TX1996: 481-501Crossref Google Scholar). The unfolding of proteins in the envelope of E. coli uniquely induces the ςE regulon but not Eς32 (3Missiakas D. Raina S. Mol. Microbiol. 1998; 28: 1059-1066Crossref PubMed Scopus (259) Google Scholar, 4Raivio T.L. Silhavy T.J. Curr. Opin. Microbiol. 1999; 2: 159-165Crossref PubMed Scopus (153) Google Scholar). ςE (RpoE) is a member of the extracytoplasmic function (ECF) subfamily of sigma factors which function as effector molecules responding to extracytoplasmic stimuli (3Missiakas D. Raina S. Mol. Microbiol. 1998; 28: 1059-1066Crossref PubMed Scopus (259) Google Scholar, 5Lonetto M. Brown K.L. Rudd K.E. Buttner M.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7573-7577Crossref PubMed Scopus (402) Google Scholar). Some microorganisms such as Streptomyces coelicolor harbor multiple ECFs that seem specialized in responding to different extracytoplasmic stimuli (5Lonetto M. Brown K.L. Rudd K.E. Buttner M.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7573-7577Crossref PubMed Scopus (402) Google Scholar, 6Kang J.G. Paget M.S. Seok Y.J. Hahn M.Y. Bae J.B. Hahn J.S. Kleanthous C. Buttner M.J. Roe J.H. EMBO J. 1999; 18: 4292-4298Crossref PubMed Scopus (216) Google Scholar). The E. coli ςE regulon is induced specifically in response to imbalanced synthesis of outer membrane proteins (7Mecsas J. Rouvière P.E. Erickson J.W. Donohue T.J. Gross C.A. Genes Dev. 1993; 7: 2618-2628Crossref PubMed Scopus (324) Google Scholar) and to misfolding of polypeptides that have been translocated across the cytoplasmic membrane (8Missiakas D. Betton J.-M. Raina S. Mol. Microbiol. 1996; 21: 871-884Crossref PubMed Scopus (292) Google Scholar).Previous work identified several genes that are transcribed by EςE (4Raivio T.L. Silhavy T.J. Curr. Opin. Microbiol. 1999; 2: 159-165Crossref PubMed Scopus (153) Google Scholar). EςE directs its own expression.rpoE is the first gene of an operon that also containsrseA, rseB, and rseC(regulator of sigma E, genes A, B, and C (9de las Peñas A. Conolly L. Gross C.A. Mol. Microbiol. 1997; 24: 3373-3386Crossref Scopus (264) Google Scholar, 10Missiakas D. Mayer M. Lemaire M. Georgopoulos C. Raina S. Mol. Microbiol. 1997; 24: 355-371Crossref PubMed Scopus (285) Google Scholar). RseA is a short hydrophobic polypeptide that integrates into the cytoplasmic membrane. The N-terminal cytoplasmic domain of RseA binds to ςE, sequestering the sigma factor from core RNA polymerase (E) (9de las Peñas A. Conolly L. Gross C.A. Mol. Microbiol. 1997; 24: 3373-3386Crossref Scopus (264) Google Scholar, 10Missiakas D. Mayer M. Lemaire M. Georgopoulos C. Raina S. Mol. Microbiol. 1997; 24: 355-371Crossref PubMed Scopus (285) Google Scholar). The C-terminal domain of RseA protrudes into the periplasm, a compartment located between the cytoplasmic and outer membranes of E. coli. The C-terminal domain of RseA interacts with RseB, a periplasmic soluble protein (9de las Peñas A. Conolly L. Gross C.A. Mol. Microbiol. 1997; 24: 3373-3386Crossref Scopus (264) Google Scholar, 10Missiakas D. Mayer M. Lemaire M. Georgopoulos C. Raina S. Mol. Microbiol. 1997; 24: 355-371Crossref PubMed Scopus (285) Google Scholar). RseB is believed to sense the concentration of misfolded polypeptides, causing RseB dissociation from RseA and liberating cytoplasmic ςE for interaction with core RNA polymerase (11Collinet B. Yuzawa H. Chen T. Herrera C. Missiakas D. J. Biol. Chem. 2000; 275: 33898-33904Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Another model suggested proteolytic cleavage of RseA in response to the accumulation of outer membrane proteins (12Ades S.E. Connolly L.E. Alba B.M. Gross C.A. Genes Dev. 1999; 13: 2449-2461Crossref PubMed Scopus (205) Google Scholar). The function of RseC, encoded by the fourth gene of the rpoEoperon, remains unknown. EςE also transcribeshtrA and fkpA, encoding a periplasmic protease (HtrA/DegP) for the removal of misfolded polypeptides (13Strauch K.L. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1576-1580Crossref PubMed Scopus (292) Google Scholar, 14Erickson J.W. Gross C.A. Genes Dev. 1989; 3: 1462-1471Crossref PubMed Scopus (291) Google Scholar) and a periplasmic peptidyl prolyl isomerase (FkpA) involved in folding envelope proteins (8Missiakas D. Betton J.-M. Raina S. Mol. Microbiol. 1996; 21: 871-884Crossref PubMed Scopus (292) Google Scholar, 15Danese P.N. Silhavy T.J. Genes Dev. 1997; 11: 1183-1193Crossref PubMed Scopus (209) Google Scholar). rpoH, encoding the transcription factor ς32 for the cytoplasmic heat shock response, is also transcribed by EςE (14Erickson J.W. Gross C.A. Genes Dev. 1989; 3: 1462-1471Crossref PubMed Scopus (291) Google Scholar).Earlier work described the isolation of rpoE knockout mutations (16Raina S. Missiakas D. Georgopoulos C. EMBO J. 1995; 14: 1043-1055Crossref PubMed Scopus (239) Google Scholar, 17Rouvière P. de las Peñas A. Mecsas J. Lu C.Z. Rudd K.E. Gross C.A. EMBO J. 1995; 14: 1032-1042Crossref PubMed Scopus (259) Google Scholar). E. coli appears to requirerpoE for viability and growth under physiological conditions, as the mutant strains cope with loss of rpoEfunction by acquiring compensatory mutations (18de las Peñas A. Connolly L. Gross C.A. J. Bacteriol. 1997; 179: 6862-6864Crossref PubMed Google Scholar). The nature of compensatory mutations as well as the number and identity of the affected genes are still unknown. Even though rpoE seems to be essential, none of the known EςE-transcribed genes (rpoH, htrA, fkpA, rseA,rseB, rseC) is required for either growth or viability of E. coli. Taken together, all previous work suggests that EςE must transcribe additional genes that are involved in the folding of envelope proteins. To identify genes that are transcribed by EςE and to approximate the size of the ςE regulon, we have used two different experimental strategies. Small DNA segments, generated by fragmentation of the E. coli chromosome, were fused to a promoterlesslacZ reporter gene carried on a single copy plasmid. Further, the λMu53-lacZ transposon was used to generate sets of random fusion between the promoterless lacZ reporter and regulatory sequences of the chromosome of E. coli. Screening of both libraries of reporter fusions in various genetic backgrounds identified 20 promoters that activated LacZ expression in a ςE-dependent manner. A hypothesis is presented to account for the essential function of the ςEregulon and to describe the role of the identified genes in responding to misfolded polypeptides within the envelope of E. coli.DISCUSSIONEςE RNA polymerase is thought to be dedicated to expressing folding catalysts that act on proteins in the bacterial envelope. Here we measured the size of the ςE regulon with two methods: two-dimensional gel electrophoresis of RpoE-induced cells and cloning of RpoE-regulated promoters. Results from both experiments as well as previous work suggest that EςEtranscribes some 43 genes. We describe here 20 new promoters that are recognized by EςE RNA polymerase. Some of the genes regulated by EςE were hitherto unknown and have been designated ecf, for extracytoplasmic encoding function. Some of the encoded gene products are located in the periplasmic space and act directly on misfolded proteins: DsbC, FkpA, HtrA, Skp, and SurA. Some other gene products are located in the bacterial cytoplasm and serve regulatory functions that coordinate the expression of the ςE regulon with environmental conditions. RpoE, RpoH, and RpoD represent components of various RNA polymerase species, whereas RseA, RseB, and RseC regulate the availability of ςE for core RNA polymerase. Several ςE-regulated gene products are involved in the synthesis of lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria. Lipopolysaccharide has been proposed to act as a cofactor for the membrane assembly of outer membrane proteins, a pathway that appears to require Skp activity (8Missiakas D. Betton J.-M. Raina S. Mol. Microbiol. 1996; 21: 871-884Crossref PubMed Scopus (292) Google Scholar). Skp has also been shown to play other roles in envelope assembly (36Schafer U. Beck K. Muller M. J. Biol. Chem. 1999; 274: 24567-24574Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). It seems noteworthy however that skp mutant cells contain increased amounts of lipopolysaccharide within the periplasm (36Schafer U. Beck K. Muller M. J. Biol. Chem. 1999; 274: 24567-24574Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). It is as if deletion of the presumed folding factor (Skp) may lead to the simultaneous accumulation of its cofactor (lipopolysaccharide). TherfaDFCL and lpxDA genes provide known components of the lipopolysaccharide biosynthetic pathway and are transcribed by EςE polymerase. In fact, the lpxD lpxA fabZgenes are regulated by two ςE-dependent promoters: one placed in front of skp (the first gene of the operon) and a second one in front of lpxD. Our preliminary results suggest that the ecfABC gene products may also be involved in the lipopolysaccharide biosynthetic pathway. 3C. Dartigalongue, D. Missiakas, and S. Raina, unpublished data. Two ςE-regulated genes encode proteins with sensory functions. MdoG is involved in coordinating cellular pressure with the biosynthesis of periplasmic membrane-derived oligosaccharides (37Kennedy E.P. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1092-1095Crossref PubMed Scopus (123) Google Scholar), whereas CutC has been postulated to be involved in copper homeostasis (38Blattner F.R. Plunkett G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (5972) Google Scholar). The requirement of these gene products for protein folding in the periplasmic space is not immediately apparent. In this and perhaps in other cases, the presence of a ςE promoter may provide growth advantages for the E. coli host which are not related to protein folding. The largest group of ςE-regulated genes encodes proteins located in the inner (NlpB, EcfD, EcfG, EcfI, and EcfL) and outer membranes (EcfK and EcfM). The precise function of these proteins remains to be established; however, it is conceivable that the membrane proteins act directly on misfolded membrane proteins and promote either polypeptide degradation or insertion into the lipid bilayer. Alternatively, membrane proteins may be involved in the transport and assembly of lipopolysaccharide into the physiological bilayer structures.Two new members of the RpoE regulon were observed to be essential: ecfE and ecfL. Because these genes appear to be transcribed by several RNA polymerases (data not shown) and have no definitive function attributed, it is impossible to draw conclusions as to why the ςE regulon is essential forE. coli growth. EcfE appears to be a member of a large group of proteases designated RIP (regulated intramembrane proteolysis). Proteases of the RIP family are needed for diverse functions such as lipid metabolism, cell differentiation, and response to unfolded proteins (39Niwa M. Sidrauski C. Kaufman R.J. Walter P. Cell. 1999; 99: 691-702Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 40Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Cell. 2000; 100: 391-398Abstract Full Text Full Text PDF PubMed Scopus (1141) Google Scholar). We are currently investigating the role of EcfE in signaling envelope stress in E. coli.In summary, the ςE regulon has evolved to control at least two cellular processes, folding of polypeptides in the bacterial envelope and biosynthesis/transport of lipopolysaccharide. Conditions that cause unfolding of polypeptides are signaled by the RseA and RseB proteins (11Collinet B. Yuzawa H. Chen T. Herrera C. Missiakas D. J. Biol. Chem. 2000; 275: 33898-33904Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). It is conceivable that the ςE regulon can sense and respond to changes in lipopolysaccharide metabolism. Our future work will address this possibility. Heat shock and other environmental stresses result in the misfolding of polypeptides in all cells. Escherichia coliresponds to the accumulation of misfolded polypeptides by activating the transcription of heat shock genes. Heat is a drastic stress that leads to protein unfolding in general and triggers two heat shock responses controlled by two distinct RNA polymerase species in E. coli 1: α2ββ′ς32 and α2ββ′ςE, Eς32, and EςE, respectively (1Gross C.A. Neidhardt F.C. Curtis R.I. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium : Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1996: 1382-1399Google Scholar, 2Missiakas D. Raina S. Georgopoulos C. Lin E.C.C. Lynch S.A. Regulation of Gene Expression in Escherichia coli. R. G. Landes Company, Austin, TX1996: 481-501Crossref Google Scholar). The unfolding of proteins in the envelope of E. coli uniquely induces the ςE regulon but not Eς32 (3Missiakas D. Raina S. Mol. Microbiol. 1998; 28: 1059-1066Crossref PubMed Scopus (259) Google Scholar, 4Raivio T.L. Silhavy T.J. Curr. Opin. Microbiol. 1999; 2: 159-165Crossref PubMed Scopus (153) Google Scholar). ςE (RpoE) is a member of the extracytoplasmic function (ECF) subfamily of sigma factors which function as effector molecules responding to extracytoplasmic stimuli (3Missiakas D. Raina S. Mol. Microbiol. 1998; 28: 1059-1066Crossref PubMed Scopus (259) Google Scholar, 5Lonetto M. Brown K.L. Rudd K.E. Buttner M.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7573-7577Crossref PubMed Scopus (402) Google Scholar). Some microorganisms such as Streptomyces coelicolor harbor multiple ECFs that seem specialized in responding to different extracytoplasmic stimuli (5Lonetto M. Brown K.L. Rudd K.E. Buttner M.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7573-7577Crossref PubMed Scopus (402) Google Scholar, 6Kang J.G. Paget M.S. Seok Y.J. Hahn M.Y. Bae J.B. Hahn J.S. Kleanthous C. Buttner M.J. Roe J.H. EMBO J. 1999; 18: 4292-4298Crossref PubMed Scopus (216) Google Scholar). The E. coli ςE regulon is induced specifically in response to imbalanced synthesis of outer membrane proteins (7Mecsas J. Rouvière P.E. Erickson J.W. Donohue T.J. Gross C.A. Genes Dev. 1993; 7: 2618-2628Crossref PubMed Scopus (324) Google Scholar) and to misfolding of polypeptides that have been translocated across the cytoplasmic membrane (8Missiakas D. Betton J.-M. Raina S. Mol. Microbiol. 1996; 21: 871-884Crossref PubMed Scopus (292) Google Scholar). Previous work identified several genes that are transcribed by EςE (4Raivio T.L. Silhavy T.J. Curr. Opin. Microbiol. 1999; 2: 159-165Crossref PubMed Scopus (153) Google Scholar). EςE directs its own expression.rpoE is the first gene of an operon that also containsrseA, rseB, and rseC(regulator of sigma E, genes A, B, and C (9de las Peñas A. Conolly L. Gross C.A. Mol. Microbiol. 1997; 24: 3373-3386Crossref Scopus (264) Google Scholar, 10Missiakas D. Mayer M. Lemaire M. Georgopoulos C. Raina S. Mol. Microbiol. 1997; 24: 355-371Crossref PubMed Scopus (285) Google Scholar). RseA is a short hydrophobic polypeptide that integrates into the cytoplasmic membrane. The N-terminal cytoplasmic domain of RseA binds to ςE, sequestering the sigma factor from core RNA polymerase (E) (9de las Peñas A. Conolly L. Gross C.A. Mol. Microbiol. 1997; 24: 3373-3386Crossref Scopus (264) Google Scholar, 10Missiakas D. Mayer M. Lemaire M. Georgopoulos C. Raina S. Mol. Microbiol. 1997; 24: 355-371Crossref PubMed Scopus (285) Google Scholar). The C-terminal domain of RseA protrudes into the periplasm, a compartment located between the cytoplasmic and outer membranes of E. coli. The C-terminal domain of RseA interacts with RseB, a periplasmic soluble protein (9de las Peñas A. Conolly L. Gross C.A. Mol. Microbiol. 1997; 24: 3373-3386Crossref Scopus (264) Google Scholar, 10Missiakas D. Mayer M. Lemaire M. Georgopoulos C. Raina S. Mol. Microbiol. 1997; 24: 355-371Crossref PubMed Scopus (285) Google Scholar). RseB is believed to sense the concentration of misfolded polypeptides, causing RseB dissociation from RseA and liberating cytoplasmic ςE for interaction with core RNA polymerase (11Collinet B. Yuzawa H. Chen T. Herrera C. Missiakas D. J. Biol. Chem. 2000; 275: 33898-33904Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Another model suggested proteolytic cleavage of RseA in response to the accumulation of outer membrane proteins (12Ades S.E. Connolly L.E. Alba B.M. Gross C.A. Genes Dev. 1999; 13: 2449-2461Crossref PubMed Scopus (205) Google Scholar). The function of RseC, encoded by the fourth gene of the rpoEoperon, remains unknown. EςE also transcribeshtrA and fkpA, encoding a periplasmic protease (HtrA/DegP) for the removal of misfolded polypeptides (13Strauch K.L. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1576-1580Crossref PubMed Scopus (292) Google Scholar, 14Erickson J.W. Gross C.A. Genes Dev. 1989; 3: 1462-1471Crossref PubMed Scopus (291) Google Scholar) and a periplasmic peptidyl prolyl isomerase (FkpA) involved in folding envelope proteins (8Missiakas D. Betton J.-M. Raina S. Mol. Microbiol. 1996; 21: 871-884Crossref PubMed Scopus (292) Google Scholar, 15Danese P.N. Silhavy T.J. Genes Dev. 1997; 11: 1183-1193Crossref PubMed Scopus (209) Google Scholar). rpoH, encoding the transcription factor ς32 for the cytoplasmic heat shock response, is also transcribed by EςE (14Erickson J.W. Gross C.A. Genes Dev. 1989; 3: 1462-1471Crossref PubMed Scopus (291) Google Scholar). Earlier work described the isolation of rpoE knockout mutations (16Raina S. Missiakas D. Georgopoulos C. EMBO J. 1995; 14: 1043-1055Crossref PubMed Scopus (239) Google Scholar, 17Rouvière P. de las Peñas A. Mecsas J. Lu C.Z. Rudd K.E. Gross C.A. EMBO J. 1995; 14: 1032-1042Crossref PubMed Scopus (259) Google Scholar). E. coli appears to requirerpoE for viability and growth under physiological conditions, as the mutant strains cope with loss of rpoEfunction by acquiring compensatory mutations (18de las Peñas A. Connolly L. Gross C.A. J. Bacteriol. 1997; 179: 6862-6864Crossref PubMed Google Scholar). The nature of compensatory mutations as well as the number and identity of the affected genes are still unknown. Even though rpoE seems to be essential, none of the known EςE-transcribed genes (rpoH, htrA, fkpA, rseA,rseB, rseC) is required for either growth or viability of E. coli. Taken together, all previous work suggests that EςE must transcribe additional genes that are involved in the folding of envelope proteins. To identify genes that are transcribed by EςE and to approximate the size of the ςE regulon, we have used two different experimental strategies. Small DNA segments, generated by fragmentation of the E. coli chromosome, were fused to a promoterlesslacZ reporter gene carried on a single copy plasmid. Further, the λMu53-lacZ transposon was used to generate sets of random fusion between the promoterless lacZ reporter and regulatory sequences of the chromosome of E. coli. Screening of both libraries of reporter fusions in various genetic backgrounds identified 20 promoters that activated LacZ expression in a ςE-dependent manner. A hypothesis is presented to account for the essential function of the ςEregulon and to describe the role of the identified genes in responding to misfolded polypeptides within the envelope of E. coli. DISCUSSIONEςE RNA polymerase is thought to be dedicated to expressing folding catalysts that act on proteins in the bacterial envelope. Here we measured the size of the ςE regulon with two methods: two-dimensional gel electrophoresis of RpoE-induced cells and cloning of RpoE-regulated promoters. Results from both experiments as well as previous work suggest that EςEtranscribes some 43 genes. We describe here 20 new promoters that are recognized by EςE RNA polymerase. Some of the genes regulated by EςE were hitherto unknown and have been designated ecf, for extracytoplasmic encoding function. Some of the encoded gene products are located in the periplasmic space and act directly on misfolded proteins: DsbC, FkpA, HtrA, Skp, and SurA. Some other gene products are located in the bacterial cytoplasm and serve regulatory functions that coordinate the expression of the ςE regulon with environmental conditions. RpoE, RpoH, and RpoD represent components of various RNA polymerase species, whereas RseA, RseB, and RseC regulate the availability of ςE for core RNA polymerase. Several ςE-regulated gene products are involved in the synthesis of lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria. Lipopolysaccharide has been proposed to act as a cofactor for the membrane assembly of outer membrane proteins, a pathway that appears to require Skp activity (8Missiakas D. Betton J.-M. Raina S. Mol. Microbiol. 1996; 21: 871-884Crossref PubMed Scopus (292) Google Scholar). Skp has also been shown to play other roles in envelope assembly (36Schafer U. Beck K. Muller M. J. Biol. Chem. 1999; 274: 24567-24574Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). It seems noteworthy however that skp mutant cells contain increased amounts of lipopolysaccharide within the periplasm (36Schafer U. Beck K. Muller M. J. Biol. Chem. 1999; 274: 24567-24574Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). It is as if deletion of the presumed folding factor (Skp) may lead to the simultaneous accumulation of its cofactor (lipopolysaccharide). TherfaDFCL and lpxDA genes provide known components of the lipopolysaccharide biosynthetic pathway and are transcribed by EςE polymerase. In fact, the lpxD lpxA fabZgenes are regulated by two ςE-dependent promoters: one placed in front of skp (the first gene of the operon) and a second one in front of lpxD. Our preliminary results suggest that the ecfABC gene products may also be involved in the lipopolysaccharide biosynthetic pathway. 3C. Dartigalongue, D. Missiakas, and S. Raina, unpublished data. Two ςE-regulated genes encode proteins with sensory functions. MdoG is involved in coordinating cellular pressure with the biosynthesis of periplasmic membrane-derived oligosaccharides (37Kennedy E.P. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1092-1095Crossref PubMed Scopus (123) Google Scholar), whereas CutC has been postulated to be involved in copper homeostasis (38Blattner F.R. Plunkett G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (5972) Google Scholar). The requirement of these gene products for protein folding in the periplasmic space is not immediately apparent. In this and perhaps in other cases, the presence of a ςE promoter may provide growth advantages for the E. coli host which are not related to protein folding. The largest group of ςE-regulated genes encodes proteins located in the inner (NlpB, EcfD, EcfG, EcfI, and EcfL) and outer membranes (EcfK and EcfM). The precise function of these proteins remains to be established; however, it is conceivable that the membrane proteins act directly on misfolded membrane proteins and promote either polypeptide degradation or insertion into the lipid bilayer. Alternatively, membrane proteins may be involved in the transport and assembly of lipopolysaccharide into the physiological bilayer structures.Two new members of the RpoE regulon were observed to be essential: ecfE and ecfL. Because these genes appear to be transcribed by several RNA polymerases (data not shown) and have no definitive function attributed, it is impossible to draw conclusions as to why the ςE regulon is essential forE. coli growth. EcfE appears to be a member of a large group of proteases designated RIP (regulated intramembrane proteolysis). Proteases of the RIP family are needed for diverse functions such as lipid metabolism, cell differentiation, and response to unfolded proteins (39Niwa M. Sidrauski C. Kaufman R.J. Walter P. Cell. 1999; 99: 691-702Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 40Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Cell. 2000; 100: 391-398Abstract Full Text Full Text PDF PubMed Scopus (1141) Google Scholar). We are currently investigating the role of EcfE in signaling envelope stress in E. coli.In summary, the ςE regulon has evolved to control at least two cellular processes, folding of polypeptides in the bacterial envelope and biosynthesis/transport of lipopolysaccharide. Conditions that cause unfolding of polypeptides are signaled by the RseA and RseB proteins (11Collinet B. Yuzawa H. Chen T. Herrera C. Missiakas D. J. Biol. Chem. 2000; 275: 33898-33904Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). It is conceivable that the ςE regulon can sense and respond to changes in lipopolysaccharide metabolism. Our future work will address this possibility. EςE RNA polymerase is thought to be dedicated to expressing folding catalysts that act on proteins in the bacterial envelope. Here we measured the size of the ςE regulon with two methods: two-dimensional gel electrophoresis of RpoE-induced cells and cloning of RpoE-regulated promoters. Results from both experiments as well as previous work suggest that EςEtranscribes some 43 genes. We describe here 20 new promoters that are recognized by EςE RNA polymerase. Some of the genes regulated by EςE were hitherto unknown and have been designated ecf, for extracytoplasmic encoding function. Some of the encoded gene products are located in the periplasmic space and act directly on misfolded proteins: DsbC, FkpA, HtrA, Skp, and SurA. Some other gene products are located in the bacterial cytoplasm and serve regulatory functions that coordinate the expression of the ςE regulon with environmental conditions. RpoE, RpoH, and RpoD represent components of various RNA polymerase species, whereas RseA, RseB, and RseC regulate the availability of ςE for core RNA polymerase. Several ςE-regulated gene products are involved in the synthesis of lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria. Lipopolysaccharide has been proposed to act as a cofactor for the membrane assembly of outer membrane proteins, a pathway that appears to require Skp activity (8Missiakas D. Betton J.-M. Raina S. Mol. Microbiol. 1996; 21: 871-884Crossref PubMed Scopus (292) Google Scholar). Skp has also been shown to play other roles in envelope assembly (36Schafer U. Beck K. Muller M. J. Biol. Chem. 1999; 274: 24567-24574Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). It seems noteworthy however that skp mutant cells contain increased amounts of lipopolysaccharide within the periplasm (36Schafer U. Beck K. Muller M. J. Biol. Chem. 1999; 274: 24567-24574Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). It is as if deletion of the presumed folding factor (Skp) may lead to the simultaneous accumulation of its cofactor (lipopolysaccharide). TherfaDFCL and lpxDA genes provide known components of the lipopolysaccharide biosynthetic pathway and are transcribed by EςE polymerase. In fact, the lpxD lpxA fabZgenes are regulated by two ςE-dependent promoters: one placed in front of skp (the first gene of the operon) and a second one in front of lpxD. Our preliminary results suggest that the ecfABC gene products may also be involved in the lipopolysaccharide biosynthetic pathway. 3C. Dartigalongue, D. Missiakas, and S. Raina, unpublished data. Two ςE-regulated genes encode proteins with sensory functions. MdoG is involved in coordinating cellular pressure with the biosynthesis of periplasmic membrane-derived oligosaccharides (37Kennedy E.P. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1092-1095Crossref PubMed Scopus (123) Google Scholar), whereas CutC has been postulated to be involved in copper homeostasis (38Blattner F.R. Plunkett G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (5972) Google Scholar). The requirement of these gene products for protein folding in the periplasmic space is not immediately apparent. In this and perhaps in other cases, the presence of a ςE promoter may provide growth advantages for the E. coli host which are not related to protein folding. The largest group of ςE-regulated genes encodes proteins located in the inner (NlpB, EcfD, EcfG, EcfI, and EcfL) and outer membranes (EcfK and EcfM). The precise function of these proteins remains to be established; however, it is conceivable that the membrane proteins act directly on misfolded membrane proteins and promote either polypeptide degradation or insertion into the lipid bilayer. Alternatively, membrane proteins may be involved in the transport and assembly of lipopolysaccharide into the physiological bilayer structures. Two new members of the RpoE regulon were observed to be essential: ecfE and ecfL. Because these genes appear to be transcribed by several RNA polymerases (data not shown) and have no definitive function attributed, it is impossible to draw conclusions as to why the ςE regulon is essential forE. coli growth. EcfE appears to be a member of a large group of proteases designated RIP (regulated intramembrane proteolysis). Proteases of the RIP family are needed for diverse functions such as lipid metabolism, cell differentiation, and response to unfolded proteins (39Niwa M. Sidrauski C. Kaufman R.J. Walter P. Cell. 1999; 99: 691-702Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 40Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Cell. 2000; 100: 391-398Abstract Full Text Full Text PDF PubMed Scopus (1141) Google Scholar). We are currently investigating the role of EcfE in signaling envelope stress in E. coli. In summary, the ςE regulon has evolved to control at least two cellular processes, folding of polypeptides in the bacterial envelope and biosynthesis/transport of lipopolysaccharide. Conditions that cause unfolding of polypeptides are signaled by the RseA and RseB proteins (11Collinet B. Yuzawa H. Chen T. Herrera C. Missiakas D. J. Biol. Chem. 2000; 275: 33898-33904Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). It is conceivable that the ςE regulon can sense and respond to changes in lipopolysaccharide metabolism. Our future work will address this possibility. We thank O. Schneewind (UCLA) for a critical review of this manuscript.