JOURNAL OF BACTERIOLOGY, May 1987, p. 2044-2049 Vol. 169, No. 5 0021-9193/87/052044-06$02.00/0 Copyright © 1987, American Society for Microbiology Feric-Coprogen Receptor FhuE of : Processing and Sequence Common to all TonB-Dependent Outer Membrane Receptor MARTIN SAUER, KLAUS HANTKE, AND VOLKMAR BRAUN* Mikrobiologie II, Universitat Tubingen, D-7400 Tubingen, Federal Republic of Germany Received 9 September 1986/Accepted 6 February 1987

Iron transport via siderophores requires outer membrane receptor proteins and the TonB . The FhuE protein of Escherichia coli functions as the receptor for ferric coprogen and ferric-rhodotorulic acid. A chromosomal DNA fragment bearing the flauE was cloned into pACYC184. The gene was localized by insertion mutagenesis by using the transposon TnlOOO. Expression in minicells revealed a FhuE precursor with an apparent molecular weight of 82,000 and a FhuE protein with a molecular weight of 76,000. The polarity of the JIuE gene was deduced from the size of truncated polypeptides derived from Tn1000 insertions, which were mapped by restriction analysis. The processing of truncated precursors that were synthesized by insertion mutants was strongly reduced even when the insertion site was close to the carboxy terminus of the FhuE protein. It is concluded that either the efficent insertion of proFhuE into the cytoplasmic membrane or the rate of cleavage of the signal peptide requires a particular conformation of the proFhuE protein, which is only formed by the complete primary structure. The amino-terminal amino acid sequence deduced from the nucleotide sequence was confirmed by gas-phase sequencing of the precursor and the mature form, which were separated by electrophoresis on polyacrylamide gels. The precursor contained an unusually long signal peptide of 36 amino acids. The amino-terminal end of the mature form contained the sequence Glu-Thr-Val-Ile-Val. A pentapeptide starting with either Glu or Asp, followed by Thr, and two uncharged residues ending with Val were found in all outer membrane receptor proteins that were constituents of TonB-dependent transport systems.

From results of studies concerning the energy requirement this sequence forms the binding domain for the TonB pro- and TonB dependence of the irreversible adsorption of tein. phages Ti and 480 to Escherichia coli cells, we concluded in Transport systems for five ferric-siderophore compounds 1976 (14) that the function determined by the tonB region have been identified in E. coli (for recent reviews, see may be to couple the energized state of the cytoplasmic references 6 and 26). One of these siderophores is coprogen membrane to the TonA outer membrane receptor protein. B, which is formed by Neurospora crassa and by certain Later it was shown that ligands did bind to their receptors in Penicillium species (20). The existence ofa specific transport unenergized cells or in tonB mutants but remained there and system for coprogen was deduced from the discovery of an were not translocated across the outer membrane (7, 18, 32). outer membrane receptor protein, designated FhuE (17). In some cases the requirement for the outer membrane Mutants in flzuE were defective in the transport of iron receptor protein for uptake could be bypassed by osmotic delivered as coprogen or as a complex with rhodotorulic shock (7) or by the preparation of spheroplasts (36). Under acid, which is another metabolite of certain fungi. In both these conditions the TonB function was no longer needed, siderophores hydroxamate groups serve as the iron-binding suggesting that it was indeed involved in the translocation of sites. Consistent with other hydroxamate siderophores (e.g., iron siderophores (12, 15), vitamin B12 (3, 32), and the aerobactin, ferrichrome), ferric coprogen and rhodotorulic B-group (31) across the outer membrane. Despite acid bind to specific outer membrane receptor proteins, this consistent evidence in favor of the original proposal, while subsequent transport steps are catalyzed by common direct proof of a physical interaction between the TonB ,flzuB,fluC,fluD, tonB, and exbB (11, 17). protein (25, 28-30) and outer membrane receptor proteins is To determine whether the FhuE protein also contains the lacking. homology region at the amino-terminal end common to all Recently, the nucleotide sequences of the structural genes TonB-dependent receptor proteins, we cloned the gene and for the receptors of vitamin B12 (19), ferric aerobactin (21), sequenced this region. Moreover, we found an unusually ferrichrome (9; R. Burkhardt, unpublished data), and ferric long signal peptide and a rather slow processing rate of the enterochelin (23) were determined. They all possess a con- FhuE precursor. sensus sequence near the amino-terminal end of the mature proteins. A mutant in vitamin B12 transport, which still MATERIALS AND METHODS adsorbed a normal amount of vitamin B12 with unaltered affinity, apparently was defective in the vectorial release of Bacterial strains and media. The E. coli K-12 strains used vitamin B12 from the receptor into the periplasm. In this in this study are listed in Table 1. For the isolation of the mutant a leucine residue was replaced by a proline residue fhuE: :A plac Mu53 mutant JB1690, strain MC4100 was within the consensus sequence (19). It was suggested that infected with X plac Mu53 (8). Neomycin-resistant colonies were isolated. These colonies were red on MacConkey plates containing 50 ,uM 2,2'-dipyridyl. To determine lacZ * Corresponding author. expression under the control of an iron-regulated operator, 2044 VOL. 169, 1987 FERRIC-COPROGEN RECEPTOR PROTEIN OF E. COLI 2045

TABLE 1. E. coli K-12 strains used in this study inserts in the JhuE gene of pKH6 were screened on NBD Source of plates, which were supplemented with 0.2 mM Fe3+- Strain Genotype reference Desferal and 40 pLg of chloramphenicol per ml. fhuE+ strains CR63 F+ Universitat formed larger colonies on Fe3+-ferrioxamine B than didffhuE Tubingen mutant strains. The plasmids were isolated from 16 conju- MC4100 F- araD139 A1acUl69 rpsL150 relAl M. Casada- gants that formed very small colonies and were designated flbBS301 deoCI ptsF25 rbsR ban MS1 to MS16. They were cleaved with the enzymes Sail, H1443 As MC4100, but aroB 17 HindlIl, PvuII, and KpnI to determine the insertion sites and H1619 As H1443, but JhuE::Mu dlX This study the orientation of TnJOOO. H1729 As H1443, but cirfhuA fiu::Mu dlX This study Tabor and Richardson (34) constructed vectors in which JB1690 As MC4100, butjhuE::X plac Mu53 This study inserted genes are transcribed by the T7 polymerase under DS410 minA minB rpsL lacY xyl thi mtl 10 the control of a T7 promoter. The plasmid pT7 6 contains a JM101 supE thi A(lac proAB) (F, traD36 37 the proAB lacIq lac ZM15) polylinker preceded by promoter of the T7 gene 10. In IR20/111 aroB cirfhuA fepA malA Universitat addition, they contain the bla gene with a transcription Tubingen polarity opposite to that of the 4i10 promoter region (S. MS44 As IR20/111, but containing pGPl-2 This study Tabor, private communication). The 6.7-kilobase (kb) and pMS44 HindIII-EcoRI fragment of pKH6 was cloned into pT7-6 MS50 As H1729, but containing pKH6 This study digested with HindIII-EcoRI. Ampicillin-resistant trans- MS172 As H1443, but containingfluE::X This study formants of E. coli H1443 were screened, and plasmids were plac Mu53 reisolated and tested for the expected insert by restriction MS200 As DS410, but containing pKH6 This study analysis. The resulting plasmid pMS44 was transferred into MS201-211 As DS410, but containing pMS1-11 This study E. coli IR20/111 which was transformed earlier by pGP1-2, WM1576 HfrC lambda carrying pGPl-2 34 which was isolated from E. coli WM1576. An ampicillin- and neomycin-resistant derivative was designated E. coli MS44. Recombinant DNA techniques. Digestion with restriction the red colonies were cross-streaked onto MacConkey plates enzymes was performed by the method described by that contained 50 ,uM Fe3+, and then onto these plates was Maniatis et al. (24) or as recommended by the supplier placed a paper strip that was impregnated with 20 mM (Boehringer GmbH, Mannheim, Federal Republic of Ger- 2,2'-dipyridyl. The dipyridyl diffused from the paper strip many). Plasmids were isolated as described by Birnboim (5). into the agar and formed a gradient of iron deficiency. DNA was treated with RNase and then with proteinase K Colonies that were red near the paper strip and white further (24). For purification of DNA by CsCl gradient centrifuga- away contained lacZ under iron regulation (16). Mutants in tion, the ethanol precipitate of DNA of a 1-liter culture was ffhuE were selected on the basis of no growth stimulation by dissolved in 2.4 ml of 10 mM Tris hydrochloride-1 mM coprogen B on iron-deficient NBD plates (8 g of nutrient EDTA (pH 7.5, TE buffer). Cesium chloride (4.2 g) and 0.4 broth, 5 g of NaCl per liter, 0.2 mM 2,2'-dipyridyl). To ml of ethidium bromide (10 mg/ml) were added. This solution exclude mutants in fhuB, fhuC, flhuD, tonB, and exbB, was layered below 8 ml of CsCl (6.4 g in 10 ml of TE buffer) growth stimulation by ferrichrome, the uptake system of and then centrifuged for 4.5 h at 65,000 x g. which shares these genes with the coprogen uptake system, DNA was sequenced by the enzymatic method (33, 37) by was tested. Mutants in fhuE were also albomycin sensitive, using [35S]dATP for labeling (4) and the M13-sequencing kit whereas Jfhu mutants have been shown to be albomycin from Amersham Buchler, Braunschweig, Federal Republic resistant (11, 26). Growth promotion was tested around filter of Germany. The DNA fragments were separated with an paper disks to which 15 pul of a 1-mg/ml solution of coprogen electrophoresis system (Macrophor 2010; LKB, Grafelfing, or ferrichrome was added (17). Insertion of A plac Mu53 into Federal Republic of Germany) (2). .fhuE was verified by the lack of the FhuE protein in gel Expression of plasmid-encoded proteins. Minicells were electrophoretograms of outer membrane proteins (17). isolated from 300 ml of an overnight culture of strain DS410, The resulting mutant JB1690 was used to construct the which was transformed with pKH6 and its TnlOOO insertion ampicillin-sensitive JhuE mutant MS172, which was aroB derivatives, in tryptone-yeast extract medium (10) supple- and therefore could not synthesize its own siderophore mented with glucose (1 mg/ml) and chloramphenicol (40 enterochelin (enterobactin). As a result it grew very slowly ,ug/ml). They were purified by three cycles of sucrose on NBD plates in the presence or absence of coprogen. gradient (10 to 30%) centrifugation. A sample (0.5 ml) An ampicillin-sensitive JhuE aroB strain was needed to adjusted to 1010 cells per ml was incubated for 1 h with 4 mM assay fragments of the flzuE region of pKH6 (see Fig. 1) that cycloserine in M9 medium. Labeling was started after 1 h at were cloned into pUC18 and pUC19 for their ability to 37°C by adding 740 kBq of [35S]methionine in 50 pAl of complement JhuE. plac Mu53 was transduced with phage methionine assay medium (Difco Laboratories, Detroit, Pl from strain JB1690 into E. coli H1443. Colonies that grew Mich.). After 1 h, cells were centrifuged, suspended in 0.05 on tryptone-yeast extract agar plates containing 50 ,ug of ml of sample buffer, heated for 5 min at 95°C, and then neomycin per ml were streaked onto MacConkey- subjected to sodium dodecyl sulfate (SDS)-polyacrylamide lactose-0.2 mM dipyridyl plates. Red colonies that were gel electrophoresis (16, 22). 35S-labeled outer membrane white on plates in which dipyridyl was replaced with 40 p,M proteins and the unlabeled proteins phosphorylase a (molec- iron ammonium sulfate contained lacZ under the control of ular weight, 93,000), human transferrin (80,000), bovine the iron-regulated ihuE operator. serum albumin (67,000), ovalbumin (45,000), and chymotryp- For insertion mutagenesis with the transposon TnlO00 sinogen (24,000) served as standards to determine the appar- (13), the F+ strain CR63 was transformed with the plasmid ent molecular weights of the proteins. The outer membrane pKH6. Transposition of TnJOOO onto pKH6 was assayed by was prepared by differential centrifugation and extraction of transferring the intermediary cointegrate between F and cytoplasmic membrane components with Triton X- pKH6 by conjugation into E. coli H1619 fhuE. Tn1000 100-MgCl2 as described previously (16). Proteins were 2046 SAUER ET AL. J. BACTERIOL.

0 1 2 3 4 5 6 7 a kb AB2847 is unable to synthesize a siderophore that could Il I I withdraw iron from the added ligands. Plasmid pKH1 from the flhuE+ clone was shortened by excising a 2-kb EcoRV m fragment. The resulting plasmid pKH6 still complemented 70I -C - _ _~~~Cwll S0a e c I I the fihuE mutation. I I -W I I III Characterization of plasmid pKH6. The restriction map of 0T 0 OZx pKH6 is shown in Fig. 1. The JhuE gene was roughly I F I = 1 1 mapped on pKH6 by insertion mutagenesis, with the trans- poson TnJOOO selecting forffliE mutants (Fig. 1). All of the

9 TnlOOO insertions were located within the 2.3-kb KpnI 9 w & - _ 0 0 0 fragment. The insertion mutants also allowed deduction of the transcriptional polarity of thefhuE gene from the deter- 000 1 mination of the size of truncated polypeptides that were Kpn Sol Kpn synthesized by the TnlOOO insertion mutants. Polypeptides expressed by pKH6 and its derivatives. The minicell-producing strain DS410 was transformed with the I I H1 t plasmid pKH6 and with the TnJOOO insertion derivatives pMS1, pMS5, pMS6, pMS10, and pMS11 (see Fig. 1 for designations). From the resulting strains, termed MS200, FIG. 1. Physical map and restriction sites of plasmid pKH6. The MS201, MS205, MS206, MS210, and MS211, respectively, thick line indicates the vector DNA ofpACYC184. The enlargement minicells were prepared and labeled with [35S]methionine. In at the bottom of the figure shows the insertion sites of transposon MS200(pKH6) three polypeptides were labeled (Fig. 2, des- TnlOOO within the large KpnI fragment. The orientation of TnlOOO ignated 200). Their electrophoretic mobilities corresponded points from 8 to -y above the line, and from -y to 8 below the line. to molecular weights of 82,000, 76,000, and 22,000. The 76,000-molecular-weight protein (76 K protein) corresponds stained with Coomassie blue before they were subjected to to the previously determined size of the mature FhuE autoradiography. protein (17). The 82K protein increased relative to the 76K Sequencing of the proFhuE and FhuE protein. For the protein when the labeling was performed in the presence of isolation of proFhuE, outer membranes were prepared from 8% ethanol. This treatment retards processing of precursor E. coli MS44. An overnight culture grown at 27°C was proteins into mature forms (27), so that the 82K protein is centrifuged, and the cells were suspended in double the likely to be the precursor of FhuE (Fig. 2, compare the lanes volume of a medium consisting of 2% tryptone-1% yeast with ethanol with those without ethanol). The 22K protein is extract-0.5% NaCl-0.2% glycerol-50 mM potassium hydro- the chloramphenicol transacetylase encoded by the vector. gen phosphate (pH 7.2). After shaking for 25 min at 42°C, 100 In Fig. 2, OM represents radioactively labeled E. coli outer ,ug ofrifampin was added per ml. The culture was shaken for membrane proteins isolated from strain AB2847 which con- 2 h at 37°C. Cells were harvested, and the outer membranes tained the plasmid pPF3-V1, a pBR322 derivative carrying were isolated as described above. the entire Jhu operon (11). It overexpresses the mature and For the isolation of the FhuE protein, outer membranes precursor forms of the FhuA (TonA) outer membrane pro- from logarithmically growing cells of E. coli MS50 were tein (both proteins are poorly resolved in Fig. 2) and, prepared. together with the porins OmpF-OmpC and the OmpA pro- The proteins of the outer membranes were separated by electrophoresis on a 1.5-mm-thick gel consisting of 8% acrylamide-0.8% bisacrylamide in the presence of 0.1% Fhu A SDS. The reagents used were of the highest purity commer- 72 72 .._.-W-_ ~~~~~._. _ cially available (BioRad, Munich, Federal Republic of Ger- many). SDS was heated twice with active carbon and was crystallized twice. After electrophoresis the proteins were transferred electrophoretically onto activated glass fiber -.; .._ .- - _ paper sheets basically as described previously (1). They _ Omp F/C were stained and the well-separated proFhuE and FhuE 4_ OmpA protein bands were cut out and sequenced directly by Edman degradation in a gas-phase sequencer (Applied 4.. Biosystems). Cat

RESULTS _ + -2 + 20+6 2 + 21+ 20 + MS 201 205 206 210 211 200 Cloning of theflauE region. Chromosomal DNA of E. coli OM AB2847 was partially digested with the restriction enzyme FIG. 2. Autoradiograph of an SDS-polyacrylamide gel Sau3A. Fragments of 5 to 10 kb were isolated from agarose electrophoretogram of [35S]methionine-labeled proteins in minicells gels and ligated into the BamHI site ofpACYC184. ThejhuE derived from strains MS201(pMS1), MS205(pMS5), MS206(pMS6), mutant H1619 fhuE::Mu dlX, which carries a temperature- MS210(pMS10), MS211(pMSll), and MS200(pKH6). Labeling was performed in the absence of (-) or the presence (+) of 8% ethanol. resistant Mu dl insertion in the JhuE gene (17), was trans- The arrows point to the precursors and processed forms of the formed with the ligation mixture. A JhuE1 derivative was truncated (pMS series) and the complete (pKH6) FhuE protein. Cat selected on NBD plates that were supplemented with 2 ,uM denotes the chloramphenicol transacetylase encoded by the vector, coprogen B. Dipyridyl and coprogen bind the iron of the 72 denotes the 72K protein encoded by Tnl000, and OM indicates medium and prevent the growth of fhuE mutants. Strain the outer membrane proteins that were used as standards. VOL. 169, 1987 FERRIC-COPROGEN RECEPTOR PROTEIN OF E. COLI 2047

10 20 30 40 50 mutants was independent of the insertion site and is un- 5'-AAAGACATIC TCGITrc ITACATGACA AATATGAAG OGTATATITC doubtedly expressed from the transposon. 60 70 80 90 100 Ethanol-treated samples of minicells carrying plasmids TATrIGCAT TrACAAACAA AAITATI ACACTAAATA TAAT¶XlCA with TnlOOO insertions contained increased amounts of 110 120 130 140 150 labeled proteins (Fig. 2). We have no explanation for this TMCAOI CCGGCAMAG CCIKACGACA ACATAAACCA AGAGATIICA because it was not observed in additional experiments. 160 170 180 However, the 190 product relationshipbasic ofconclusionthe larger regardingand smallerthepolypeptidesprecursor- GAT and the slow processing rate which is more pronounced in AetMet LeuCeru SerThAThr GinCMePheATAsn Arg Asp AsnMn GinCn ¶IyrTr GnGin AaAla Iele the insertion mutants is not affected. 200 210 220 230 240 Cloning of KpnI fragments into the plasmids pUC18 and ACC AM CCG TCA CA CIT GCC GGrT TC ATA GCA CI=J GCA CIA ITA pUC19. All ofthefluE-negative mutants that were generated Thr Lys Pro Ser Leu Leu Ala Gly Cys Ile Ala Leu Ala Leu Leu by TnJOOO insertion occurred within a 2.3-kb KpnI fragment 250 260 270 280 (Fig. 1). To see whether the entire JhuE gene was contained OXT TCT GCCC MT2 GCrTIGCA cCA GCC ACr GAA GAA ACG GTG AT on this fragment, it was excised and cloned into pUC18 and Pro Ser Ala Ala Phe Ala Ala Pro Ala Thr Glu Glu Thr Val Ile pUC19. E. coli JM101 was transformed, and white colonies 290 300 310 320 330 devoid of a-complementation of ,-galactosidase were scored GIT GAG GrT TCA GMC ACA GCr OCA GAT GAT Cr;C GAA AAT GAT TAC on plates containing isopropyl thiogalactoside and 5-bromo- Val Glu Gly Ser Ala Thr Ala Pro Asp Asp Gly Glu Asn Asp Tyr 4-chloro-3-indolyl-fi-D-galactopyranoside. The plasmids 340 350 were isolated and cut with KpnI to determine the size of the theinsert.0.6-kbPlasmidsKpnIcarryingfragmentsthe 2.3-kbin pUC18as wellandaspUC19the 2.3- plus AGC GTA ACG TCTACeThrGCeA G G ACC-3' were Ser Val Thr Ser Thr Ser Ala Gly TIhxr obtained. None of the plasmids could complement E. coli FIG. 3. Nucleotide sequence of the upstream region and the MS172 JfluE::X plac Mu53 (Table 1) to fhuE'. This shows amino-terminal end of the JhuE gene. The underlined hexanucleo- that the complete JhuE gene is not contained on either the tide is recognized by the endonuclease KpnI. The arrow marks the 2.3- or the 2.9-kb fragment. The structural gene had to site at which the signal peptidase cleaves the FhuE precursor extend beyond the leftmost KpnI site because cleavage of protein. the promoter region alone would have been compensated for by the lac promoter of pUC18 or pUC19, which contained teins, served as standards in this experiment to determine the inserts in both orientations. the apparent size of the plasmid-encoded polypeptides. To support the conclusion presented above, the 3' end of Labeled minicell proteins were also compared with unla- the structural gene was sequenced. The 0.7-kb HindIII-KpnI beled stained standard proteins (see above). fragment of pKH6 was cloned into phage M13 mpl9, and the Strain MS211 contained a TnlOOO insertion distal to the fragment was sequenced in the direction of KpnI to HindIII vector (Fig. 1). It expressed two proteins, the sizes of which which resulted in a polynucleotide of 358 base pairs (Fig. 3). could not be distinguished from those synthesized by MS200 Synthetic oligonucleotides complementary to the sequences (Fig. 2). Strain MS210 formed two proteins (81K and 72K) between base pairs 1 and 21 and base pairs 218 and 235 were that were smaller. The 72K protein comigrated with the used to sequence the complementary strand. The sequence TnlOOO-encoded 72K protein (13). A pair of proteins was contains an open reading frame that indeed starts 201 base also formed by strains MS206, MS205, and MS201 with pairs in front of the left KpnI site. The open reading frame is molecular weights of 66,000 and 57,000; 57,000 and 49,000; preceded by a possible ribosome binding site 5'-AGAGAT-3' and 29,000 and 23,000, respectively. The declining size of the five nucleotides in front of the start codon. Nucleotides 89 to proteins of MS211 to MS201 corresponded to the TnlOOO 94 form an ideal -10 promoter region 5'-TATAAT-3'. Tran- insertion sites in pKH6 and demonstrate that they are scription could start at the adenine -residue in the sequence truncated forms of the FhuE protein. The transcription 5'-CAT-3' of nucleotides 99 to 101. The -35 region is deduced from this set of FhuE derivatives proceeds from left probably located at nucleotides 60 to 65 anA consists of the to right (Fig. 1). sequence 5'-TTTACA-3'. The concentration of the precursor of the FhuE protein Determination of the amino-terminal amino acid sequence was rather high even without the addition of ethanol. After of the precursor and the mature form. To confirm the amino TnlOOO insertion the amount of the precursors was even acid sequence deduced from the nucleotide sequence and to higher. It seems that overproduced FhuE is rather slowly determine the cleavage site of the signal peptidase, the processed in minicells and that the rate of processing de- precursor and the mature form of FhuE were isolated, To creases when the carboxy-terminal end of FhuE is deleted. avoid contamination of proFhuE and FhuE with iron- The presence of a 39K protein in all TnJOOO insertion regulated outer membrane proteins of similar electrophoretic

FhuE Ala Pro Ala Thr GluP9T hVTat-ViT-TVaT1GIu Gly Ser Ala FhuA Ala Val Glu Pro Lys Glul Asp Thr Ile Thr ValiThr Ala Ala Pro BtUB Gln Asp Thr Ser Asp Thr Leu Val Val 'Thr Ala Asn Arg Pro: -I lut Gln Gln Thr Asp Asp Glu Thr Phe Val VaI 'Ser Ala Asn Arg FepA Gln Glu Pro Thr Asp Thr Pro Val Ser His Asp' Asp Thr Ile Val VaI ,Thr Ala Ala Glu FIG. 4. Amino-terminal amino acid sequences of the TonB-dependent outer membrane receptor proteins FhuE, FhuA, BtuB, lut, and FepA. The invariant amino acids threonine and valine and the leucine residue, which is replaced by a proline residue in the btuB451 mutant, are underlined. The cleavage sites of the FhuE and FhuA precursors are the only ones that have been confirmed by determination ofthe amino acid sequences at the amino-terminal ends of the mature proteins. 2048 SAUER ET AL. J. BACTERIOL. mobility, the plasmid-encoded IhuE gene was expressed in very different. The other four transposon insertion mutants strains that were lacking the three proteins Cir, FhuA, FepA also exhibited a similarly low rate ofprocessing. We propose and Fiu, respectively (Table 1). Synthesis of proFhuE was that the removal of the carboxy-terminal end of the proFhuE strongly enhanced by cloningfluE downstream of the strong protein affects cleavage of the signal peptide. It appears that T7 promoter 4i10 (data not shown). Transcription was con- the processing rate is dependent on a certain conformation of fined to JhuE by inactivation of the temperature-sensitive proFhuE that is only formed by the complete primary repressor of the T7 polymerase at 42°C and by inhibition of structure. This particular conformation may determine the the E. coli polymerase with rifampin. After electrophoresis rate of insertion of the proFhuE protein into the cytoplasmic the proteins were transferred by electroblotting from the membrane, or the rate of hydrolysis at the signal peptide polyacrylamide gel onto glass filter paper. The proFhuE or cleavage site. In contrast, processing ofthe proFhuA protein FhuE protein band-containing glass filter paper strip was was faster under similar experimental conditions, and no placed directly into a gas-phase sequencer. The phenylthio- decrease in rate was observed for truncated proFhuA pro- hydantoin derivatives of the amino acids released by Edman teins (11). The FhuE protein may well be an example of degradation were identified by high-pressure liquid chroma- posttranslational processing. tography. The sequence obtained for the precursor was The FhuE protein contains the consensus sequence (Fig. Met-Leu-Ser-Thr-Gln; the sequence obtained for the mature 4), which is apparently typical of outer membrane receptor protein was Ala-Pro-Ala-Thr-Glu-Glu-Thr-Val. proteins, the function of which is dependent on the TonB function. The invariant amino acids threonine and valine DISCUSSION (underlined in Fig. 4) are surrounded by homologous amino acids at the same positions in the different receptor proteins, The complementation assays with TnlOOO insertion mu- for example, aspartate and glutamate, valine, leucine and tants apd KpnI fragments of pKH6 localized the thuE gene isoleucine, glycine and alanine, serine and threonine. The on pKH6. The polynucleotide sequence indeed revealed an TonB protein (29) was found in both the cytoplasmic (28) and open reading frame that started upstream of the left KpnI site the outer membrane (R. J. Kadner, personal communica- (Fig. 1 and Fig. 3). The sequences of the ribosome binding tion). This arrangement is compatible with the notion that site and the -10 promoter region are identical to those in the TonB protein forms intermembrane bridges and regu- front of the .fuA gene (9; R. Burkhardt, unpublished data). lates the activity of outer membrane proteins according to The amino acid sequence contains a sequence of about 17 the energy state of the cytoplasmic membrane. hydrophobic amino acids that is preceded by a more hydrophilic sequence containing the two positively charged ACKNOWLEDGMENTS amino acids arginine and lysine. These are typical features of signal peptides, although they are usually only about 22 We thank F. Lottspeich for the amino acid sequence analysis, R. Gross and W. Eisenbeiss for a preliminary restriction analysis of amino acids long; and the positively charged amino acids are plasmid pKH1, and K. Poole for comments on the manuscript. concentrated closer to the amino-terminal methionine resi- This study was supported by the Deutsche Forschungsgemein- due (35). The sequence also contains a typical cleavage site schaft (SFB 323). for the signal peptidase (Fig. 3, arrow). Proforms of proteins that are entirely or partially translocated across the cytoplas- LITERATURE CITED mic membrane frequently possess the sequence Ala-X-Ala- 1. Aebersold, R. H., D. B. Teplow, L. E. Hood, and S. B. H. Kent. Ala at the cleavage site, where X is any amino acid; this 1986. Electroblotting onto activated glass. J. Biol. Chem. 261: sequence is also present in the proFhuA protein. Determi- 4229-4238. nation of the amino-terminal sequences of the precursor and 2. Ansorge, W., and R. Barker. 1984. System for DNA sequencing the mature FhuE protein confirmed cleavage at this position with resolution up to 600 base pairs. J. Biochem. Biophys. rather than at the additional Ala-Ala or Methods 9:33-47. Ala-Leu site, which 3. Bassford, P. J., Jr., C. Bradbeer, R. J. Kadner, and C. A. is nearer to the amino-terminal end. As a result the proFhuE Schiraitman. 1976. Transport of vitamin B12 in tonB mutants of protein carries an unusually long signal peptide composed of Escherichia coli. J. Bacteriol. 128:242-247. 36 amino acids, which is somewhat larger than that of the 4. Biggin, M. D., T. J. Gibson, and G. F. Hong. 1983. Buffer FhuA protein (33 amino acids). This conclusion is supported gradient gels and 35S-label as an aid to rapid DNA sequence by the two forms of FhuE that are synthesized in minicells determination. Proc. Natl. Acad. Sci. USA 80:3963-3965. (Fig. 2). The difference in molecular weight determined by 5. Birnboim, H. D. 1983. A rapid alkaline extraction method for the SDS-polyacrylamide gel electrophoresis amounts to about isolation of plasmid DNA. Methods Enzymol. 100:243-255. 6,000; the calculated value for the signal peptide is 3,856. 6. Braun, V. 1985. The iron transport systems of Escherichia coli, The difference between the calculated and the determined p. 617-652. In A. N. Martenosi (ed.), The enzymes of biological membranes, vol. 3. Plenum Publishing Corp., New York. size is larger than the accuracy limits of gel electrophoresis 7. Braun, V., S. Frenz, K. Hantke, and K. Schafler. 1980. Penetra- can account for. It may be that one of the two proteins tion of M into cells of Escherichia coli. J. Bacteriol. 142: behaves anomalously on polyacrylamide gels; perhaps it is 162-168. the proFhuE protein with its large hydrophobic signal pep- 8. Bremer, E., T. J. Silhavy, and G. Weinstock. 1985. Transposable tide. X plac Mu for creating lacZ operon fusions and An interesting observation is the rather slow processing of kanamycin resistance insertions in Escherichia coli. J. Bacte- the precursor into the mature form. This was reproducibly riol. 162:1092-1099. observed in minicells programmed by the complete f7huE 9. Coulton, J. W., P. Mason, D. R. Cameron, G. Cannel, R. Jean, gene. The rate of processing was strongly reduced in strain and H. N. Rode. 1986. Protein fusions of 0-galactosidase to the ferrichrom-iron receptor of E. coli K-12. J. Bacteriol. 165:181- MS211, with TnlOOO insertion being close to the carboxy- 192. terminal end (Fig. 2, compare 211 [strain MS2111] with 200 10. Dougan, G., and D. Sherratt. 1977. The transposon Tnl as a [strain MS200]). Although the difference in size between the probe for studying ColEl structure and function. Mol. Gen. complete and the truncated FhuE protein was too small to be Genet. 151:151-166. resolved by gel electrophoresis, the conversion rates were 11. Fecker, L., and V. Braun. 1983. Cloning and expression of the VOL. 169, 1987 FERRIC-COPROGEN RECEPTOR PROTEIN OF E. COLI 2049

Afu genes involved in iron(III)-hydroxamate uptake by Esche- cloning: a laboratory manual. Cold Spring Harbor Laboratory, richia coli. J. Bacteriol. 156:1301-1314. Cold Spring Harbor, N.Y. 12. Frost, G. E., and H. Rosenberg. 1975. Relationship between the 25. Mann, B. J., C. D. Holroyd, C. Bradbeer, and R. J. Kadner. tonB locus and iron transport in Escherichia coli. J. Bacteriol. 1986. Reduced activity of TonB-dependent functions in strains 124:704-712. of Escherichia coli. FEMS Microbiol. Lett. 33:255-260. 13. Guyer, M. S. 1983. Uses of the transposon y8 in the analysis of 26. Neilands, J. B. 1981. Microbial iron compounds. Annu. Rev. cloned genes. Methods Enzymol. 101:362-369. Biochem. 50:715-731. 14. Hancock, R. E. W., and V. Braun. 1976. Nature of the energy 27. Palva, T. E., T. R. Hirst, S. J. S. Hardy, and J. Randall. 1981. requirement for the irreversible adsorption ofbacteriophages Ti Synthesis of a precursor to the B subunit of heat-labile and +80 to Escherichia coli. J. Bacteriol. 125:409-415. enterotoxin in Escherichia coli. J. Bacteriol. 146:325-330. 15. Hancock, R. E. W., and V. Braun. 1977. Iron transport in 28. Plastow, G. S., and J. B. Holland. 1979. Identification of Escherichia coli K-12: 2,3 dihydroxybenzoate-promoted iron Escherichia coli inner membrane polypeptide specified by a uptake. Arch. Microbiol. 114:231-239. X-tonB transducing . Biochem. Biophys. Res. 16. Hantke, K. 1981. Regulation of ferric iron transport in Esche- Commun. 90:1007-1014. richia coli K-12. Isolation of a constitutive mutant. Mol. Gen. 29. Postle, K., and R. E. Good. 1983. DNA sequence of the Genet. 182:288-292. Escherichia coli tonB gene. Proc. Natl. Acad. Sci. USA 80: 17. Hantke, K. 1983. Identification of an iron uptake system specific 5235-5239. for coprogen and rhodotorulic acid in Escherichia coli K-12. 30. Postle, K., and W. S. Reznikoff. 1979. Identification of the Mol. Gen. Genet. 191:301-306. Escherichia coli tonB gene product in minicells containing tonB 18. Hantke, K., and V. Braun. 1978. Functional interaction of the hybrid plasmids. J. Mol. Biol. 131:619-636. tonA/tonB receptor system in Escherichia coli. J. Bacteriol. 31. Pugsley, A. P., and P. Reeves. 1976. Characterization of group B 135:190-197. colicin-resistant mutants ofEscherichia coli K-12: colicin resist- 19. Heller, K., and R. J. Kadner. 1985. Nucleotide sequence of the ance and the role of enterochelin. J. Bacteriol. 127:218-228. gene for the vitamin B12 receptor protein in the outer membrane 32. Reynolds, P. R., G. P. Mottur, and C. Bradbeer. 1980. Transport of Escherichia coli. J. Bacteriol. 161:904-908. of vitamin B12 in Escherichia coli. Some observations on the 20. Keller-Schierlein, W., and H. Diekmann. 1970. Zur Konstitution roles of the gene products of BtuC and TonB. J. Biol. Chem. des Coprogens. Helv. Chim. Acta 53:2035-2044. 255:4313-4319. 21. Krone, W. J. A., F. Stegehuis, G. Koningstein, C. van Doorn, B. 33. Sanger, F., A. R. Coulson, B. G. Barrel, A. J. H. Smith, and Roosendaal, F. K. de Graaf, and B. Oudega. 1985. Character- B. A. Roe. 1980. Cloning in single-stranded bacteriophage as an ization of the pColV-K30 encoded cloacin DF13/aerobactin aid to rapid DNA sequencing. J. Mol. Biol. 143:161-178. outer of Escherichia coli, isolation and 34. Tabor, S., and C. C. Richardson. 1985. A bacteriophage T7 purification of the protein and analysis of its nucleotide se- RNA polymerase/promoter system for controlled exclusive ex- quence and primary structure. FEMS Microbiol. Lett. 26: pression of specific genes. Proc. Natl. Acad. Sci. USA 82:1074- 153-161. 1078. 22. Lugtenberg, G., J. Mejers, R. Peters, P. van der Hoek, and L. 35. van Heine, G. 1985. Signal sequences. The limits ofvariation. J. van Alphen. 1975. Electrophoretic resolution of the major outer Mol. Biol. 184:99-105. membrane protein of Escherichia coli K-12 into four bands. 36. Weaver, C. A., and J. Konisky. 1980. tonB-independent fer- FEBS Lett. 58:254-258. richrome-mediated iron transport in Escherichia coli sphero- 23. Lundrigan, M. D., and R. J. Kadner. 1986. Nucleotide sequence plasts. J. Bacteriol. 143:1513-1518. of the gene for the ferrienterochelin receptor FepA in Esche- 37. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved richia coli: homology among outer membrane receptors which M13 phage cloning vectors and host strains: nucleotide se- interact with TonB. J. Biol. Chem. 261:10797-10801. quence of the M13mpl8 and pUC19 vectors. Gene 33:113- 24. Maniatis, T., E. F. Fritsch, and H. Sambrook. 1982. Molecular 119.