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• W2022732685 abstract "We report a novel outer membrane lipoprotein of Escherichia coli. DNA sequencing between ampC and sugE at the 94.5 min region of the E. coli chromosome revealed an open reading frame specifying 177 amino acid residues. Primer extension analysis demonstrated that the promoter is activated at the transition between exponential and stationary growth phases under control of the rpoS sigma factor gene, and this was confirmed in vivo by monitoring expression of β-galactosidase activity from a lacZ translational fusion. The amino acid sequence exhibited 31% identity with human apolipoprotein D (apoD), which is a component of plasma high density lipoprotein and belongs to the eukaryotic family of lipocalins. The bacterial lipocalin (Blc) contained a short deletion of 7 amino acid residues corresponding to a hydrophobic surface loop that is thought to facilitate the physical interaction between apoD and high density lipoprotein. However, Blc exhibited a typical prokaryotic lipoprotein signal peptide at its amino terminus. Overexpression, membrane fractionation, and metabolic labeling with [3H]palmitate demonstrated that Blc is indeed a globomycin-sensitive outer membrane lipoprotein. Blc represents the first bacterial member of the family of lipocalins and may serve a starvation response function in E. coli. We report a novel outer membrane lipoprotein of Escherichia coli. DNA sequencing between ampC and sugE at the 94.5 min region of the E. coli chromosome revealed an open reading frame specifying 177 amino acid residues. Primer extension analysis demonstrated that the promoter is activated at the transition between exponential and stationary growth phases under control of the rpoS sigma factor gene, and this was confirmed in vivo by monitoring expression of β-galactosidase activity from a lacZ translational fusion. The amino acid sequence exhibited 31% identity with human apolipoprotein D (apoD), which is a component of plasma high density lipoprotein and belongs to the eukaryotic family of lipocalins. The bacterial lipocalin (Blc) contained a short deletion of 7 amino acid residues corresponding to a hydrophobic surface loop that is thought to facilitate the physical interaction between apoD and high density lipoprotein. However, Blc exhibited a typical prokaryotic lipoprotein signal peptide at its amino terminus. Overexpression, membrane fractionation, and metabolic labeling with [3H]palmitate demonstrated that Blc is indeed a globomycin-sensitive outer membrane lipoprotein. Blc represents the first bacterial member of the family of lipocalins and may serve a starvation response function in E. coli. INTRODUCTIONThe lipocalin superfamily consists of widely distributed, primarily extracellular, eukaryotic proteins that bind and transport small hydrophobic ligands(1Flower D.R. North A.C.T. Attwood T.K. Protein Sci. 1993; 2: 753-761Google Scholar). The molecular structures of four lipocalins (plasma retinol-binding protein(2Newcomer M.E. Jones T.A. Åqvist J. Sundelin J. Eriksson U. Rask L. Peterson P.A. EMBO J. 1984; 3: 1451-1454Google Scholar), bilin-binding protein (3Huber R. Schneider M. Mayr I. Müller R. Deutzmann R. Suter F. Zuber H. Falk H. Kayser H. J. Mol. Biol. 1987; 198: 499-513Google Scholar), insecticyanin(4Holden H.M. Rypniewski W.R. Law J.H. Rayment I. EMBO J. 1987; 6: 1565-1570Google Scholar), and α-lactalbumin(5Papiz M.Z. Sawyer L. Eliopoulos E.E. North A.C.T. Findlay J.B.C. Sivaprasadarao R. Jones T.A. Newcomer M.E. Kraulis P.J. Nature. 1986; 324: 383-385Google Scholar)), revealed a common structural motif that consists of an eight-stranded antiparallel β-barrel, arranged as two stacked orthogonal sheets, with a COOH-terminal α-helix. Despite the common lipocalin fold, only 25-30% amino acid sequence identity exists between lipocalins of known structure(6Godovac-Zimmermann J. Trends Biochem. Sci. 1988; 13: 64-66Google Scholar). The cup-shaped three-dimensional structure of the lipocalins, which forms a central hydrophobic binding pocket for the ligand, is also characteristic of the fatty acid-binding proteins; these represent another recently identified protein family that also binds small hydrophobic molecules, but which by contrast, contain a 10-stranded antiparallel β-barrel and are almost exclusively intracellular. Because of their similarities of structure and function, Flower and co-workers (1Flower D.R. North A.C.T. Attwood T.K. Protein Sci. 1993; 2: 753-761Google Scholar) have proposed the classification of lipocalins and fatty acid-binding proteins into a larger structural superfamily termed calycins.Although lipocalins are generally soluble proteins, apolipoprotein D (apoD) 1The abbreviations used are: apoDapolipoprotein DHDLhigh density lipoproteinBlcbacterial lipocalinLBLuria-Bertani mediumPAGEpolyacrylamide gel electrophoresisPCRpolymerase chain reactionIPTGisopropyl-1-thio-β-D-galactopyranoside. was originally identified as a component of the plasma high density lipoprotein (HDL) particle, leading to the suggestion that apoD may transport a component of the lecithin-cholesterol acyltransferase reaction(7Drayna D. Fielding C. McLean J. Baer B. Castro G. Chen E. Comstock L. Henzel W. Kohr W. Rhee L. Wion K. Lawn R. J. Biol. Chem. 1986; 261: 16535-16539Google Scholar). Unlike the classical apolipoproteins, which are embedded in the lipoprotein surface by extended amphipathic α-helical structures, homology modeling of apoD against the atomic coordinates of bilin binding protein suggested that apoD associates with the HDL particle by a hydrophobic surface loop. This modeling study also postulated that a heme-related compound may be the preferred ligand for apoD(8Peitsch M.C. Boguski M.S. New Biol. 1990; 2: 197-206Google Scholar). However, apoD has also been identified as a progesterone- and pregnenolone-binding protein isolated from breast fluid, suggesting a role in the transport of steroid hormones in human mammary tissue(9Balbin M. Freije J.M.P. Fueyo A. Sánchez L.M. López-Otín C. Biochem. J. 1990; 271: 803-807Google Scholar). In the cyst fluid of women with gross cystic disease of the breast, apoD can exceed the concentration found in plasma by about 1000-fold(10Pearlman W.H. Gueriguian J.L. Sawyer M.E. J. Biol. Chem. 1973; 248: 5736-5741Google Scholar), and apoD induction by both retinoic acid (11López-Boado Y.S. Tolivia J. López-Otín C. J. Biol. Chem. 1994; 269: 26871-26878Google Scholar) and interleukin-1-α (12Blais Y. Sugimoto K. Carriere M.C. Haagensen D.E. Labrie F. Simard J. Int. J. Cancer. 1994; 59: 400-407Google Scholar) has been demonstrated in human breast cancer cells, suggesting that apoD may be a marker of hormonal alterations. Additionally, apoD accumulates in regenerating and remyelinating peripheral nerve, suggesting a role in lipid transport within extravascular compartments(13Boyles J.K. Notterpek L.M. Anderson L.J. J. Biol. Chem. 1990; 265: 17805-17815Google Scholar, 14Spreyer P. Schaal H. Kuhn G. Rothe T. Unterbeck A. Olek K. Müller H.W. EMBO J. 1990; 9: 2479-2484Google Scholar). Like other members of the lipocalin superfamily, apoD appears to be able to transport a variety of ligands in a number of different contexts.Despite the presence of lipocalins in a wide range of eukaryotic organisms, no lipocalin has ever been identified in bacteria(15Sansom C.E. North A.C.T. Sawyer L. Biochim. Biophys. Acta. 1994; 1208: 247-255Google Scholar). Additionally, the apolipoprotein components of plasma lipoproteins are unrelated to bacterial lipoproteins, which are anchored to membranes by a lipid-modified amino-terminal cysteine residue(16Braun V. Wu H.C. Ghuysen J.-M. Hackenbeck R. Bacterial Cell Wall: New Comprehensive Biochemistry. Vol. 27. Elsevier Science Publishers B.V., Amsterdam1993: 319-342Google Scholar). In this report, we describe an outer membrane lipoprotein of Escherichia coli, which is clearly homologous to apoD. This protein, which we term Blc (bacterial lipocalin), is encoded by the blc gene at 94.5 min on the E. coli chromosome, immediately downstream of the ampC β-lactamase operon. The blc promoter is expressed at the onset of stationary growth phase under control of the rpoS sigma factor gene, which directs expression of genes necessary for adaptation to starvation conditions. Blc is the first lipocalin identified in a bacterial species and may provide an evolutionary link between bacterial and plasma lipoproteins.EXPERIMENTAL PROCEDURESBacterial Strains and Growth ConditionsE. coli MC4100 (F-, Δ[argF-lac]U169, araD139, rpsL150, ptsF25, flbB5301, rbsR, deoC, relA1) was provided by G. Cecchini (17Westenberg D.J. Gunsalus R.P. Ackrell B.A.C. Sices H. Cecchini G. J. Biol. Chem. 1993; 268: 815-822Google Scholar), and RH90 (MC4100, rpoS359::Tn10) was provided by R. Hengge-Aronis(18Lange R. Hengge-Aronis R. J. Bacteriol. 1991; 173: 4474-4481Google Scholar). E. coli TG1 supE, hsdΔ5, thi, Δ(lac-proAB), F‘[traD36, proAB+, lacIq, lacZΔM15] was obtained from Amersham Corp. Bacteria were grown aerobically in a New Brunswick shaker at 37°C in Luria Bertani (LB) medium(19Miller J.H. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992Google Scholar). Strains harboring plasmids or transposons were supplemented appropriately with antibiotics (ampicillin at 100 μg/ml, chloramphenicol at 42.5 μg/ml, and tetracycline at 12.5 μg/ml). Overnight cultures were subcultured at a 1% inoculum and growth monitored with a Klett-Summerson colorimeter.MaterialsDNA polymerase from Thermus aquaticus and restriction enzymes were obtained from Life Technologies, Inc. Reverse transcriptase from avian myeloblastosis virus, RNase A, bacteriophage T4 polynucleotide kinase, and glycogen were obtained from Boehringer Mannheim, whereas RNAguard was obtained from Pharmacia Biotech Inc. [9,10-3H]Palmitate (54 Ci/mmol) was obtained from Amersham Buchler, whereas [γ-32P]ATP (6 000 Ci/mmol) was obtained from DuPont. Globomycin was a gift from Sankyo Co., Ltd., Tokyo. 4-Methylumbelliferone and 4-methylumbelliferyl-β-D-galactoside were obtained from Sigma. All other materials were obtained from commercial sources.DNA and Protein AnalysisPlasmid purifications, restriction enzyme digestions, ligations, transformations, and DNA electrophoresis were performed according to Sambrook et al.(20Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Electroporation was performed with a Cell-Porator (Life Technologies, Inc.) according to the manufacturers instructions. DNA synthesis and sequencing was performed in the Core Facility of the Department of Biochemistry, University of Alberta using an Applied Biosystems model 392 DNA/RNA synthesizer and model 373A DNA sequencer (Perkin Elmer). Protein was determined according to Lowry(21Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Google Scholar), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Bishop and Weiner(22Bishop R.E. Weiner J.H. Eur. J. Biochem. 1993; 213: 405-412Google Scholar).DNA Sequencing of the blc Gene from E. coliA 6.1-kilobase EcoRI fragment, corresponding to the 94.5 min region of the chromosome from E. coli strain CS520, was subcloned from plasmid pLC16-43 (23Clarke L. Carbon J. Cell. 1976; 9: 91-99Google Scholar) into pACYC184(19Miller J.H. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992Google Scholar). The resulting plasmid (pAmpAC) was propagated in E. coli MC4100 and used as a double-stranded template for sequencing both strands of the genomic region between ampC(24Jaurin B. Grundström T. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4897-4901Google Scholar) and sugE(25Greener T. Govezensky D. Zamir A. EMBO J. 1993; 12: 889-896Google Scholar). The forward primer (EcPst: 5′-GTCGTTGCCTGCAGTTCTCC-3′) hybridizes between nucleotides 148 and 167 (Fig. 1), whereas the reverse primer (EcSma: 5′-AACTACCAGGCTGC TGTACC-3′) hybridizes between nucleotides 615 and 634 (Fig. 1).Transcriptional Mapping of the E. coli blc PromoterRNA was prepared by the modified hot phenol extraction method of Frost et al.(26Frost L. Lee S. Yanchar N. Paranchych W. Mol. & Gen. Genet. 1989; 218: 152-160Google Scholar) and resuspended in diethylpyrocarbonate-treated water at 10 mg/ml. The primer used in primer extension analysis (EcBlc: 5′-GGAGAACTGCAGGCAACGACC-3′) hybridizes between nucleotides 147 and 167 (Fig. 1). Total cellular RNA (20 μg) was annealed to 0.5 pmol of primer, labeled at the 5′ end with [γ-32P]ATP using T4 polynucleotide kinase(19Miller J.H. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992Google Scholar). The primer and RNA were mixed in a 30-μl volume of annealing buffer (3 M NaCl, 0.5 M Tris-HCl, pH 7.5, and 1 mM EDTA, pH 8.0), denatured at 85°C for 5 min, and allowed to anneal at 37°C for a minimum of 1 h. Following annealing, the nucleic acids were precipitated with ethanol and the pellets allowed to air dry before they were resuspended in a 25-μl volume of reverse transcriptase buffer containing 0.5 mM dNTPs and 15 units of RNAguard. Reverse transcriptase (20 units) was added, and the reaction was incubated at 42°C for 1 h. RNA was removed by treatment with RNase A for 15 min at 37°C, and the DNA was precipitated with ethanol in the presence of 0.3 M sodium acetate, pH 4.8, and 10 μg of glycogen. The products were separated on a 6% sequencing gel alongside dideoxy sequencing reactions using the same primer as was used for the extension reaction. Gels were dried, and autoradiography was carried out at −70°C for 3 weeks.Construction and Analysis of a blc::lacZ Translational Fusion PlasmidA 4-kilobase EcoRI/KpnI fragment from pAmpAC was cloned into pLacZY2 (27Jain C. Gene (Amst.). 1993; 133: 99-102Google Scholar) to create a translational fusion plasmid (pBlcLacEK) in which the first 42 codons of blc are followed by lacZ. A 1.8-kilobase BamHI/ClaI fragment from pMF5 (28Mulvey M.R. Loewen P.C. Nucleic Acids Res. 1989; 17: 9979-9991Google Scholar) was cloned into pACYC184 to create pACKatF, which carries a functional allele of rpoS on a vector that is compatible with pLacZY2 and pBlcLacEK; these latter two plasmids could only be transformed into E. coli MC4100 and RH90 by electroporation. β-Galactosidase activity was monitored fluorometrically using 4-methylumbelliferyl-β-D-galactoside as substrate according to the method of Youngman(29Youngman P. Hardy K.G. Plasmids: A Practical Approach. IRL Press, Oxford, UK1987: 79-102Google Scholar), except that activities were normalized against total protein assayed (in milligrams) rather than the optical densities of the cultures. Measurements were determined with a Sequoia-Turner model 450 fluorometer equipped with NB360 and SC430 filters.Construction of an Inducible Blc Expression PlasmidAmplification of blc from E. coli by the polymerase chain reaction (PCR) was performed according to Bishop and Weiner (22Bishop R.E. Weiner J.H. Eur. J. Biochem. 1993; 213: 405-412Google Scholar) using the forward primer (EcEco: 5′-ATATGAATTCAAGAGGAAACATTTATGCGCC-3′) for which the 18 residues at the 3′ end hybridize between nucleotides 92 and 109 (Fig. 1); the 17 residues at the 3′ end of the reverse primer (EcHind: 5′-TATAAAGCTTTAACTACCAGGCTGCTG-3′) hybridize between nucleotides 619 and 635 (Fig. 1). PCR was performed through 25 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s, using pAmpAC as template. The PCR product was digested with EcoRI and HindIII for cloning into expression vectors pMS119EH and pMS119HE (30Strack B. Lessl M. Calendar R. Lanka E. J. Biol. Chem. 1992; 267: 13062-13072Google Scholar) digested with the same restriction enzymes; the resulting plasmids were designated pBlcEH and pBlcHE, respectively, and differ only in the orientation of the blc gene with respect to the inducible tac promoter. The blc gene cloned behind the tac promoter (pBlcEH) was subjected to double-stranded sequencing as indicated above using the same primers for the PCR amplification.Membrane Isolation and FractionationE. coli MC4100 (250 ml) harboring pBlcEH or pBlcHE was grown for 2 h and induced with 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) for an additional 2 h before harvesting. Cells were resuspended in 10 ml of ice-cold Buffer A (50 mM sodium phosphate, pH 7.1, 10 mM MgCl2), adjusted to 10 μg/ml in DNase, and mechanically disrupted by passage through a precooled French press cell at 1000 p.s.i. Unbroken cells were removed by low speed centrifugation at 4°C before the membranes were sedimented at 90,000 rpm in a Beckman TL 100.3 rotor for 30 min. Samples of the supernatant and membrane fractions, as well as the whole cell lysates, were analyzed by SDS-PAGE. The membranes were resuspended in 200 μl of ice-cold Buffer A and layered on top of a discontinuous sucrose gradient composed of 0.5 ml of 60%, 1 ml of 55%, 2.4 ml of 50%, 2.5 ml of 45%, 2.4 ml of 40%, 1.4 ml of 35%, and 0.8 ml of 30% sucrose in Buffer A. Centrifugation was at 4°C for 16 h at 35,000 × g in a Beckman SW41 rotor(31Walderich B. Ursinus-Wössner A. van Duin J. Höltje J.-V. J. Bacteriol. 1988; 170: 5027-5033Google Scholar). The separated membranes were harvested from the centrifuge tube and analyzed by SDS-PAGE; sucrose was removed by trichloroacetate precipitation as described by Bishop and Weiner(22Bishop R.E. Weiner J.H. Eur. J. Biochem. 1993; 213: 405-412Google Scholar).Palmitate Labeling of Induced ProteinFor palmitate labeling, 25-ml cultures of E. coli MC4100 harboring pBlcEH or pBlcHE were grown for 1 h, and 10 ml were transferred into growth flasks containing 50 μCi of [3H]palmitate from which toluene had been evaporated over a gentle stream of N2(g). The cultures were made 1 mM in IPTG, 100 μg/ml in ampicillin and, when necessary, 10 μg/ml in globomycin. Cells were grown an additional 1 h, harvested, washed once with 50 mM sodium phosphate, pH 7.2, and resuspended in 120 μl of 20 mM Tris-HCl, pH 8, containing 1 mM EDTA and 1% SDS. Cells were incubated for 5 min at 100°C and centrifuged at 14,000 × g for 10 min. The supernatant (100 μl) was added to 1 ml of ice-cold acetone and kept overnight at 4°C before centrifugation as above. The pellet was thoroughly resuspended in 50 μl of 1% SDS, and an aliquot corresponding to about 800,000 cpm was separated by 15% SDS-PAGE and the labeled protein visualized by fluorography. Relative molecular mass was estimated using Rainbow low molecular mass standards (Amersham Corp.).Computer MethodsSequence alignments were performed using the GCG (Genetics Computer Group, University of Wisconsin) FASTA and TFASTA algorithms. Protein structure was analyzed using FRODO (32Jones T.A. Methods Enzymol. 1985; 115: 157-171Google Scholar) and MOLSCRIPT(33Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Google Scholar).RESULTSSequence Analysis of the blc GeneApproximately 370 base pairs of DNA that remained to be characterized between the 3′ end of ampC and the 3′ end of sugE, which is oriented toward ampC in the 94.5 min region of the E. coli chromosome, was subjected to DNA sequencing. From the published sequence of ampC(24Jaurin B. Grundström T. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4897-4901Google Scholar), we designed a forward sequencing primer (EcPst; see “Experimental Procedures”), whereas the published sequence of sugE(25Greener T. Govezensky D. Zamir A. EMBO J. 1993; 12: 889-896Google Scholar) allowed us to design a reverse sequencing primer (EcSma; see “Experimental Procedures”). By utilizing the genomic DNA from E. coli CS520 carried on plasmid pAmpAC as a double-stranded template, it was possible to unambiguously assign the sequence of both DNA strands between ampC and sugE. The 660 base pairs that separate ampC and sugE in E. coli CS520 are shown in Fig. 1.A single open reading frame was revealed, which specified 177 amino acid residues (19,853 Da) and exhibited a consensus prokaryotic lipoprotein cleavage site (16Braun V. Wu H.C. Ghuysen J.-M. Hackenbeck R. Bacterial Cell Wall: New Comprehensive Biochemistry. Vol. 27. Elsevier Science Publishers B.V., Amsterdam1993: 319-342Google Scholar) predicting a mature protein of 159 residues (18,043 Da). The initiating methionine codon was separated from the rhoindependent terminator of the ampC gene by 60 base pairs, from which −35 and −10 hexamers corresponding weakly to an E. coli σ-70 promoter could be distinguished. A reasonable ribosome binding sequence was appropriately positioned upstream of the initiating methionine codon. The open reading frame converged upon sugE such that the two genes shared overlapping translational termination codons; no rhoindependent terminator structure could be discerned between them. A chromosomal deletion in E. coli MI1443 encompasses the blc locus; this strain is capable of growth under both aerobic (34Bishop R.E. Weiner J.H. FEMS Microbiol. Lett. 1993; 114: 349-354Google Scholar) and anaerobic (35Condon C. Weiner J.H. Mol. Microbiol. 1988; 2: 43-52Google Scholar) conditions, indicating that the blc gene is dispensable in E. coli.While this manuscript was in preparation, the DNA sequence of the E. coli MG1655 chromosomal region from 92.8-0.1 min was submitted from the E. coli genome project under GenBank accession number U14003. The sequence from E. coli MG1655 confirms that determined by us for E. coli CS520, except for a C to G transversion at nucleotide 498; this is a silent mutation in the third position of glycine codon 132. We also determined the sequence of the blc homolog from Citrobacter freundii OS60; this DNA sequence, together with neighboring loci, will be described elsewhere. Pertinent to this study was the identification of an open reading frame specifying 177 amino acid residues and displaying 90% amino acid sequence identity with E. coli Blc.Transcriptional Mapping of the E. coli blc PromoterTo verify that the blc gene was expressed, primer extension analysis was performed using the primer EcBlc (see “Experimental Procedures”). A weakly expressed cDNA corresponding to a transcript whose 5′ end mapped to nucleotide 80 in Fig. 1 was detected from E. coli MC4100 (Fig. 2). Autoradiography was performed for 3 weeks in order to detect this cDNA. The expression of the blc mRNA was improved if the cells were allowed to grow into stationary phase.Figure 2:Transcriptional mapping and rpoS control of the blc promoter. The deduced sequence of the blc promoter region derived from the sequencing lanes labeled G, A, T, and C is shown vertically on the left. The primer extension reactions were performed with total cellular RNA isolated from E. coli strains MC4100 (rpoS+) and RH90 (MC4100, rpoS359:: Tn10). RNA samples were isolated at mid-exponential (1), late exponential (2), early stationary (3), and stationary (4) growth phases, which were reached after 200, 270, 340, and 540 min of growth, respectively. The position of the cDNA corresponding to the blc transcription start site (+1) is marked by an arrowhead.View Large Image Figure ViewerDownload (PPT)E. coli MC4100 was recently reported to be distinct from many other common strains of E. coli by virtue of having a functional allele of rpoS, which controls a program of gene expression induced under starvation conditions and at the onset of stationary phase(36Kaasen I. Falkenberg P. Stryvold O.B Str⊘m A.R. J. Bacteriol. 1992; 174: 889-898Google Scholar, 37Hengge-Aronis R. Cell. 1993; 72: 165-168Google Scholar). In order to determine if the accumulation of the blc transcript in stationary phase was determined by rpoS, we also performed primer extension analysis using E. coli RH90 (MC4100 rpoS359::Tn10). The blc transcript was not detected in E. coli RH90 under all observed growth phases (Fig. 2), indicating that the blc gene belongs to the stationary phase regulon controlled by rpoS, and suggesting that the Blc protein may serve a function that contributes to the adaptation of cells to starvation conditions. Although there is no clearly defined consensus sequence for rpoS-dependent promoters, they are generally similar in structure to those controlled by σ-70(38Tanaka K. Takayanagi Y. Fujita N. Ishihama A. Takahashi H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3511-3515Google Scholar, 39Nguyen L.H. Jensen D.B. Thompson N.E. Gentry D.R. Burgess R.R. Biochemistry. 1993; 32: 11112-11117Google Scholar).Monitoring Activity of a blc::lacZ Translational Fusion in VivoTo determine if the primer extension results were valid in vivo, a blc::lacZ translational fusion was created from pLacZY2, which was developed to exhibit low background β-galactosidase activity, thus permitting the analysis of weakly expressed genes(27Jain C. Gene (Amst.). 1993; 133: 99-102Google Scholar). The results (Table 1) demonstrate that activities expressed from the fusion plasmid (pBlcLacEK) were only significantly above those expressed from pLacZY2 when a functional rpoS allele was present, indicating that σ-70 has no detectable effect on the blc promoter. A slight 1.5-fold increase above background activity was determined in exponential phase cultures (200 min of growth) in the presence of the chromosomal rpoS gene from E. coli MC4100, and this was matched by a 1.4-fold increase in the presence of the plasmid encoded rpoS gene (pACKatF) harbored by E. coli RH90. However, in stationary phase cultures (overnight growth), the chromosomal rpoS gene provided a 2.5-fold increase above background activity, whereas the plasmid encoded rpoS gene provided a 5.3-fold increase. These results confirm that blc is normally expressed only very weakly in E. coli and that this expression is optimized in stationary phase under the control of rpoS.Tabled 1 Open table in a new tab Controlled Expression of Blc ProteinTo express the Blc protein, the blc gene from E. coli was cloned behind the IPTG-inducible tac promoter in the expression vector pMS119EH to create pBlcEH. The blc gene was also cloned in the reverse orientation in pMS119HE to create pBlcHE, which serves as a negative control. The gene was amplified by PCR using primers designed to incorporate an EcoRI restriction enzyme site and improved ribosome binding sequence at the 5′ end (EcEco; see “Experimental Procedures”) and a HindIII restriction enzyme site at the 3′ end (EcHind; see “Experimental Procedures”). The DNA sequence of the PCR product in pBlcEH was identical to that of the template, except for a G to A transition at nucleotide 516; this is a silent mutation in the third position of leucine codon 138. Blc was induced in E. coli MC4100 and protein divided into cellular lysate, soluble, and membrane fractions. Samples analyzed by SDS-PAGE are shown in Fig. 3. A doublet of bands migrating with molecular masses of 16 and 18 kDa in the Blc expressing lanes were visible in the cellular lysate and the membrane fractions. This finding is consistent with an accumulation of a membrane protein, which is being processed to a mature species by the removal of an 18-amino acid signal peptide and the addition of 700 Da, the approximate molecular mass attributed to lipid modification of prokaryotic lipoproteins. The two unique bands in the Blc expressing lanes migrate slightly faster than their expected positions of 18 and 20 kDa for the mature and precursor species of Blc, respectively. This anomolous migration may result from lipid modification of Blc, which would promote binding of SDS and facilitate migration in SDS-PAGE.Figure 3:Overexpression and membrane localization of the Blc protein. 12% SDS-PAGE analysis of E. coli MC4100 transformed with either pBlcEH, expressing blc (+), or pBlcHE, unable to express blc(-). Cells were induced with IPTG and subjected to French pressure lysis. The lysates (L) were divided into soluble (S) and membrane (M) fractions by ultracentrifugation. The position of bands present during Blc expression are marked by arrowheads. Each lane corresponds to 40 μg of protein stained with Coomassie Blue dye. The indicated molecular mass standards are expressed in kDa.View Large Image Figure ViewerDownload (PPT)Subcellular Localization of Blc ProteinTo localize the two Blc species in the Gram-negative cell envelope of E. coli, membranes were fractionated by sucrose density gradient centrifugation. Light, heavy, and medium fractions were harvested; these represent the cytoplasmic membrane, outer membrane, and adhesion zones between outer and cytoplasmic membranes, respectively(31Walderich B. Ursinus-Wössner A. van Duin J. Höltje J.-V. J. Bacteriol. 1988; 170: 5027-5033Google Scholar). The band corresponding to the Blc precursor was localized in the cytoplasmic membrane fraction, whereas the band corresponding to mature Blc was localized in the outer membrane fraction, and both species were equally distributed among the fraction of adhesion zones (Fig. 4). This result suggests that the Blc precursor accumulates in the cytoplasmic membrane, and that mature Blc is targeted to the outer membrane.Figure 4:Membrane fractionation of precursor and mature species of Blc. 15% SDS-PAGE analysis of E. coli MC4100 transformed with either pBlcEH, expressing blc (+), or pBlcHE, unable to express blc(-). Cells were induced with IPTG and subjected to French pressure lysis. Membranes were isolated by ultracentrifugation and separated into light (L), medium (M), and heavy (H) fractions by sucrose density gradient centrifugation. The pBlcEH fractions shown at right are aligned to show the precursor and mature forms of Blc. The position of bands present during" @default.
• W2022732685 created "2016-06-24" @default.
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• W2022732685 creator A5009092288 @default.
• W2022732685 creator A5012993374 @default.
• W2022732685 creator A5026785330 @default.
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• W2022732685 date "1995-09-01" @default.
• W2022732685 modified "2023-10-16" @default.
• W2022732685 title "Stationary Phase Expression of a Novel Escherichia coli Outer Membrane Lipoprotein and Its Relationship with Mammalian Apolipoprotein D" @default.
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