Sequence of the fhuE outer-membrane receptor gene of Escherichia coli K12 and properties of mutants

The fhuE gene of Escherichia coli codes for an outer-membrane receptor protein required for the uptake of iron(III) via coprogen, ferrioxamine B and rhodotorulic acid. The amino acid sequence, deduced from the nucleotide sequence, consisted of 729 residues. The mature form, composed of 693 residues, has a calculated molecular weight of 77,453, which agrees with the molecular weight of 76,000 determined by polyacrylamide gel electrophoresis. The FhuE protein contains four regions of homology with other TonB-dependent receptors. A valine to proline exchange in the 'TonB box' abolished transport activity. Phenotypic revertants with substitutions of arginine, glutamine, or leucine at the valine position exhibited increasing iron-coprogen transport rates. Point mutations resulting in the replacement of glycine (127) in the second homology region with either alanine, aspartate, valine, asparagine or histidine exhibited decreased transport rates (listed in descending order). A truncated FhuE protein lacking 24 amino acids at the C-terminal end was exported to the periplasm but failed to be inserted into the outer membrane.     

A lipoprotein of Yersinia enterocolitica facilitates ferrioxamine uptake in Escherichia coli.

A cloned fragment of Yersinia enterocolitica DNA complemented the defect in ferrioxamine B uptake of an Escherichia coli fhuE mutant lacking the outer membrane high-affinity transport protein FhuE. Subcloning revealed that a 13.7-kDa outer membrane protein was required for complementation. The amino acid sequence deduced from the nucleotide sequence showed extensive homology to PCPHi, an outer membrane lipoprotein of Haemophilus influenzae. We therefore termed this protein PCPYe. Plasmid-encoded pcpY mediated a low-affinity uptake of ferrioxamine B which may be caused by changes in the permeability of the outer membrane due to an overexpression of this outer membrane protein. A transposon insertion mutant in the plasmid-encoded pcpY gene was transferred into the chromosome of Y. enterocolitica. The resulting mutation had no effect on the high-affinity uptake of ferrioxamine B in Yersinia cells. Using the antibiotic ferrimycin we were able to isolate a Y. enterocolitica mutant lacking the high-affinity outer membrane receptor for ferrioxamine uptake, termed FoxA.     

Localization of functional domains in the Escherichia coli coprogen receptor FhuE and the Pseudomonas putida ferric-pseudobactin 358 receptor PupA

Transport of ferric-siderophores across the outer membrane of gram-negative bacteria is mediated by specific outer membrane receptors. To localize the substrate-binding domain of the ferric-pseudobactin 358 receptor, PupA, of Pseudomonas putida WCS358, we constructed chimeric receptors in which different domains of PupA were replaced by the corresponding domains of the related ferric-pseudobactin receptors PupB and PupX, or the coprogen receptor FhuE of Escherichia coli. None of the chimeric proteins composed of pseudobactin receptor domains facilitated growth on any of the original substrates, or they showed only an extremely low efficiency. However, these receptors enabled cells of Pseudomonas BN8 to grow on media supplemented with uncharacterized siderophore preparations. These siderophore preparations were isolated from the culture supernatant of WCS358 cells carrying plasmids that contain genes of Pseudomonas B10 required for the biosynthesis of pseudobactin B10. Hybrid proteins that contained at least the amino-terminal 516 amino acids of mature FhuE were active as a receptor for coprogen and interacted with the E. coli TonB protein. A chimeric PupA-FhuE protein, containing the amino-terminal 94 amino acids of mature PupA, was also active as a coprogen receptor, but only in the presence of Pseudomonas TonB. It is concluded that the carboxy-terminal domain of ferric-pseudobactin receptors is important, but not sufficient, for ligand interaction, whereas binding of coprogen by the FhuE receptor is not dependent on this domain. Apparently, the ligand-binding sites of different receptors are located in different regions of the proteins. Furthermore, species-specific TonB binding by the PupA receptor is dependent on the amino-terminal domain of the receptor.     

Localization of functional domains in the Escherichia coli coprogen receptor FhuE and the Pseudomonas putida ferric-pseudobactin 358 receptor PupA

Transport of ferric-siderophores across the outer membrane of gram-negative bacteria is mediated by specific outer membrane receptors. To localize the substrate-binding domain of the ferric-pseudobactin 358 receptor, PupA, of Pseudomonas putida WCS358, we constructed chimeric receptors in which different domains of PupA were replaced by the corresponding domains of the related ferric-pseudobactin receptors PupB and PupX, or the coprogen receptor FhuE of Escherichia coli. None of the chimeric proteins composed of pseudobactin receptor domains facilitated growth on any of the original substrates, or they showed only an extremely low efficiency. However, these receptors enabled cells of Pseudomonas BN8 to grow on media supplemented with uncharacterized siderophore preparations. These siderophore preparations were isolated from the culture supernatant of WCS358 cells carrying plasmids that contain genes of Pseudomonas B10 required for the biosynthesis of pseudobactin B10. Hybrid proteins that contained at least the amino-terminal 516 amino acids of mature FhuE were active as a receptor for coprogen and interacted with the E. coli TonB protein. A chimeric PupA-FhuE protein, containing the amino-terminal 94 amino acids of mature PupA, was also active as a coprogen receptor, but only in the presence of Pseudomonas TonB. It is concluded that the carboxy-terminal domain of ferric-pseudobactin receptors is important, but not sufficient, for ligand interaction, whereas binding of coprogen by the FhuE receptor is not dependent on this domain. Apparently, the ligand-binding sites of different receptors are located in different regions of the proteins. Furthermore, species-specific TonB binding by the PupA receptor is dependent on the amino-terminal domain of the receptor.     

Requirements for substrate recognition by bacterial leader peptidase.

Many secreted and membrane proteins have amino-terminal leader peptides which are essential for their insertion across the membrane bilayer. These precursor proteins, whether from prokaryotic or eukaryotic sources, can be processed to their mature forms in vitro by bacterial leader peptidase. While different leader peptides have shared features, they do not share a unique sequence at the cleavage site. To examine the requirements for substrate recognition by leader peptidase, we have truncated M13 procoat, a membrane protein precursor, from both the amino- and carboxy-terminal ends with specific proteases or chemical cleavage agents. The fragments isolated from these reactions were assayed as substrates for leader peptidase. A 16 amino acid residue peptide which spans the leader peptidase cleavage site is accurately cleaved. Neither the basic amino-terminal region nor most of the hydrophobic central region of the leader peptide are essential for accurate cleavage.     

Nucleotide sequence of the gene for the ferrienterochelin receptor FepA in Escherichia coli. Homology among outer membrane receptors that interact with TonB

We have determined the nucleotide sequence of the Escherichia coli fepA gene, which codes for the outer membrane receptor for ferrienterochelin and colicins B and D. The predicted FepA polypeptide has a molecular weight of 79,908 and consists of 723 amino acids. A 22-amino acid leader or signal peptide preceded the mature protein. With respect to overall composition, hydropathy, net charge and distribution of nonpolar segments, the FepA polypeptide was typical of other E. coli outer membrane proteins, except that FepA contained 2 cysteine residues. Comparison of the deduced amino acid sequence of FepA with that of three other TonB-dependent receptors (BtuB, FhuA, and IutA) revealed only a few regions of sequence homology; one of these included the amino-termini. An amino acid substitution within the conserved amino-terminal region of BtuB resulted in production of a receptor that had normal binding functions but was incapable of energy-dependent transport of vitamin B12. This result suggests that the amino-terminal end of these four polypeptides is involved in interaction with the TonB protein or another step of energy transduction. Three other regions of homology were shared among the four proteins: one near residues 50 to 70, another at about residue 100 to 140, and the last between 20 and 40 amino acid residues from the carboxyl terminus. The function of these three regions remains speculative.     

Characterization of the Lactococcus lactis nisin A operon genes nisP, encoding a subtilisin-like serine protease involved in precursor processing, and nisR, encoding a regulatory protein involved in nisin biosynthesis.

Biosynthesis of the lantibiotic peptide nisin by Lactococcus lactis NIZO R5 relies on the presence of the conjugative transposon Tn5276 in the chromosome. A 12-kb DNA fragment of Tn5276 including the nisA gene and about 10 kb of downstream DNA was cloned in L. lactis, resulting in the production of an extracellular nisin precursor peptide. This peptide reacted with antibodies against either nisin A or the synthetic leader peptide, suggesting that it consisted of a fully modified nisin with the nisin leader sequence still attached to it. This structure was confirmed by N-terminal sequencing and 1H-nuclear magnetic resonance analysis of the purified peptide. Deletion studies showed that the nisR gene is essential for the production of this intermediate. The deduced amino acid sequence of the nisR gene product indicated that the protein belongs to the family of two-component regulators. The deduced amino acid sequence of NisP, the putative product of the gene upstream of nisR, showed an N-terminal signal sequence, a catalytic domain with a high degree of similarity to those of subtilisin-like serine proteases, and a putative C-terminal membrane anchor. Cell extracts of Escherichia coli overexpressing nisP were able to cleave the nisin precursor peptide, producing active, mature nisin. A similar activation was obtained with whole cells but not with membrane-free extracts of L. lactis strains carrying Tn5276 in which the nisA gene had been inactivated. The results indicate that the penultimate step in nisin biosynthesis is secretion of precursor nisin without cleavage of the leader peptide, whereas the last step is the cleavage of the leader peptide sequence from the fully maturated nisin peptide.     

Identification of an iron uptake system specific for coprogen and rhodotorulic acid in Escherichia coli K12

With the lac operon fusion technique, mutants were isolated in two genes that specify two outer membrane proteins designated FhuE (76 K) and Fiu (83 K). The synthesis of both proteins was increased under low iron growth conditions. The FhuE-protein was shown to be necessary for iron uptake via coprogen, an iron chelator produced by certain fungi, e.g. Neurospora crassa. In addition to fhueE the genes fhuCDB, tonB and exbB were necessary for iron coprogen uptake. The gene fhuE was mapped between kdp and gltA near 16 min on the genetic map of E. coli K12, while gene fiu was mapped near 18 min between chlA and chlE. Nor iron transport system could be assigned as yet to the Fiu protein.     

Identification and characterization of the TolC protein, an outer membrane protein from Escherichia coli

We used the cloned tolC gene to identify, locate, and purify its gene product. Strains carrying pPR13 or pPR42 overproduced a cell envelope protein (molecular weight, 52,000). A protein of the same molecular weight was identified in radioactively labeled minicells carrying pPR13; this protein was absent in pPR11-carrying minicells. This protein was the tolC gene product, since pPR11 differed from pPR13 in having a Tn10 insertion in the tolC gene. The protein seen in cell envelopes of whole cells (TolC protein) was found to exist in an aggregated state in the outer membrane; under conditions in which OmpC and OmpF were peptidoglycan associated, TolC protein was not likewise associated. Using these properties, we purified the TolC protein and determined the sequence of twelve amino acids from the amino-terminal end. The location of the TolC protein in the outer membrane was consistent with the proposed function for the tolC gene product as a processing protein in the outer membrane.     

Analysis of two gene regions involved in the expression of the imipenem-specific, outer membrane porin protein OprD of Pseudomonas aeruginosa

A Tn501 mutant of Pseudomonas aeruginosa resistant to imipenem and lacking the imipenem-specific outer membrane porin protein OprD was isolated. The mutation could be complemented to imipenem susceptibility and OprD-sufficiency by a cloned 6-kb EcoRI-PstI fragment of DNA from the region of chromosome of the wild-type strain surrounding the site of Tn501 insertion. However, this fragment did not contain the oprD structural gene as judged by its inability to hybridize with an oligonucleotide corresponding to the N-terminal amino acid sequence of OprD. DNA sequencing of 3.9 kb of the region surrounding the Tn501 insertion site revealed three large open reading frames, one of which would be interrupted by the Tn501 insertion in the mutant. This latter open reading frame, named opdE (for putative regulator of oprD expression), predicted a hydrophobic protein of M(r) 41,592. Using the above-mentioned oligonucleotide, the oprD structural gene was cloned and expressed in Escherichia coli on a 2.1-kb Bam HI-KpnI fragment. DNA sequencing predicted a 420 amino acid mature OprD protein with a 23 amino acid signal sequence.     

Rise and fall of the delta globin gene

The complete nucleotide sequence of the gene phoE, which codes for the phosphate limitation inducible outer membrane pore protein of Escherichia coli K12 was established. The results show that PhoE protein is synthesized in a precursor form with a 21 amino acid residue amino-terminal extension. This peptide has the general characteristics of a signal sequence. The promoter region of phoE has no homlogy with the consensus sequence of E. coli promoter regions, but homologous sequences with the promoter region of phoA, the structural gene for alkaline phosphatase, were observed. The deduced amino acid sequence showed that the mature PhoE protein is composed of 330 amino acid residues with a calculated molecular weight of 36,782. A number of 81 charged amino acids was found scattered throughout the protein while no large stretches of hydrophobic amino acids were observed. Hydrophobicity and hydration profiles of PhoE protein showed five pronounced hydrophilic maxima which are all located in the region from the amino terminus to residue 212.When the deduced amino acid sequence of PhoE protein was compared with the established sequence of the OmpF pore protein, a number of 210 identical residues was found. Some aspects of the structure-function relationship of PhoE protein are discussed in view of the hydrophobicity and hydration profiles, and the homology between PhoE protein and OmpF protein.     

Cloning of the Pseudomonas aeruginosa outer membrane porin protein P gene: evidence for a linked region of DNA homology.

The gene encoding the outer membrane phosphate-selective porin protein P from Pseudomonas aeruginosa was cloned into Escherichia coli. The protein product was expressed and transported to the outer membrane of an E. coli phoE mutant and assembled into functional trimers. Expression of a product of the correct molecular weight was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, using polyclonal antibodies to protein P monomer and trimer forms. Protein P trimers were partially purified from the E. coli clone and shown to form channels with the same conductance as those formed by protein P from P. aeruginosa. The location and orientation of the protein P-encoding (oprP) gene on the cloned DNA was identified by three methods: (i) mapping the insertion point of transposon Tn501 in a previously isolated P. aeruginosa protein P-deficient mutant; (ii) hybridization of restriction fragments from the cloned DNA to an oligonucleotide pool synthesized on the basis of the amino-terminal protein sequence of protein P; and (iii) fusion of a PstI fragment of the cloned DNA to the amino terminus of the beta-galactosidase gene of pUC8, producing a fusion protein that contained protein P-antigenic epitopes. Structural analysis of the cloned DNA and P. aeruginosa chromosomal DNA revealed the presence of two adjacent PstI fragments which cross-hybridized, suggesting a possible gene duplication. The P-related (PR) region hybridized to the oligonucleotide pool described above. When the PstI fragment which contained the PR region was fused to the beta-galactosidase gene of pUC8, a fusion protein was produced which reacted with a protein P-specific antiserum. However, the restriction endonuclease patterns of the PR region and the oprP gene differed significantly beyond the amino-terminal one-third of the two genes.     

Cloning and expression of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli.

The transport of cyanocobalamin (vitamin B12) in cells of Escherichia coli is dependent on a receptor protein (BtuB protein) located in the outer membrane. A 9.1-kilobase pair BamHI fragment carrying the btuB gene was cloned from a specialized transducing phage into multicopy plasmids. Insertions of transposon Tn1000 which prevented production of the receptor localized btuB to a 2-kilobase pair region. Further subcloning allowed isolation of this region as a 2.3-kilobase pair Sau3A fragment. The BtuB+ plasmids were shown by maxicell analysis to encode a polypeptide with a molecular weight of 66,000 in the outer membrane. This polypeptide was missing in cells with Tn1000 insertions in btuB and was reduced in amount upon growth of plasmid-bearing cells in repressing concentrations of vitamin B12. Several Tn1000 insertions outside the 5' end of the coding region exhibited reduced production of receptor. A deletion at the 3' end of btuB resulted in formation of an altered receptor. Amplified production of this polypeptide was associated with increased levels of binding of the receptor's ligands (vitamin B12 and phage BF23), increased rates of vitamin B12 uptake, and altered susceptibility to the group E colicins. Deficiency in various major outer membrane proteins did not affect production of the btuB product, and the amplified levels of this protein partially reversed the tolerance to E colicins seen in these mutants.     

Cloning of the gene coding for the outer membrane receptor protein for ferric pseudobactin, a siderophore from a plant growth-promoting Pseudomonas strain

Plant growth-promoting Pseudomonas B10 produces its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin under iron-limiting conditions. A structural gene encoding the 85,000-Da putative outer membrane receptor protein for ferric pseudobactin was identified in a gene bank from Pseudomonas B10 prepared with the broad host-range conjugative cosmid cloning vector pLAFR1. Transposon Tn5 mutagenesis of recombinant plasmid pJLM300 localized the functional gene to a region of approximately 2.4 kilobases consistent with the apparent molecular weight of the receptor protein. Mobilization of pJLM300 into Pseudomonas A124 and A225, whose growth was inhibited by Pseudomonas B10 or pseudobactin, rendered these strains no longer susceptible to iron starvation by pseudobactin because they were now able to transport ferric pseudobactin. Pseudobactin biosynthetic genes flanked this receptor gene on both sides and were on separate operons. Transposon Tn5 insertion mutants of Pseudomonas B10 lacking this receptor protein were generated by a marker exchange technique and were defective in ferric pseudobactin transport. Such mutants could be complemented in trans by pJLM300. The production of pseudobactin, the receptor protein, and four other outer membrane proteins in Pseudomonas B10 was coordinately regulated by the level of intracellular iron.     

Lethal mutations in the structural gene of an outer membrane protein (OmpA) of Escherichia coli K12

Summary The gene ompA encodes a major outer membrane protein of Escherichia coli. Localized mutagenesis of the part of the gene corresponding to the 21-residue signal sequence and the first 45 residues of the protein resulted in alterations which caused cell lysis when expressed. DNA sequence analyses revealed that in one mutant type the last CO₂H-terminal residue of the signal sequence, alanine, was replaced by valine. The proteolytic removal of the signal peptide was much delayed and most of the unprocessed precursor protein was fractioned with the outer membrane. However, this precursor was completely soluble in sodium lauryl sarcosinate which does not solubilize the OmpA protein or fragments thereof present in the outer membrane. Synthesis of the mutant protein did not inhibit processing of the OmpA or OmpF proteins. In the other mutant type, multiple mutational alterations had occurred leading to four amino acid substitutions in the signal sequence and two affecting the first two residues of the mature protein. A reduced rate of processing could not be clearly demonstrated. Membrane fractionation suggested that small amounts of this precursor were associated with the plasma membrane but synthesis of this mutant protein also did not inhibit processing of the wild-type OmpA or OmpF proteins. Several lines of evidence left no doubt that the mature, mutant protein is stably incorporated into the outer membrane. It is suggested that the presence, in the outer membrane, of the mutant precursor protein in the former case, or of the mutant protein in the latter case perturbs the membrane architecture enough to cause cell death.     

Characterization of genes required for pilus expression in Pseudomonas syringae pathovar phaseolicola.

Nonpiliated, phage phi 6-resistant mutants of Pseudomonas syringae pv. phaseolicola were generated by Tn5 transposon mutagenesis. A P. syringae pv. phaseolicola LR700 cosmid library was screened with Tn5-containing EcoRI fragments cloned from nonpiliated mutants. The cosmid clone pVK253 complemented the nonpiliated mutant strain HB2.5. A 3.8-kb sequenced region spanning the Tn5 insertion site contained four open reading frames. The transposon-inactivated gene, designated pilP, is 525 bp long, potentially encoding a 19.1-kDa protein precursor that contains a typical membrane lipoprotein leader sequence. Generation of single mutations in each of the three remaining complete open reading frames by marker exchange also resulted in a nonpiliated phenotype. Expression of this gene region by the T7 expression system in Escherichia coli resulted in four polypeptides of approximately 39, 26, 23, and 18 kDa, in agreement with the sizes of the open reading frames. The three genes upstream of pilP were designated pilM (39 kDa), pilN (23 kDa), and pilO (26 kDa). The processing of the PilP precursor into its mature form was shown to be inhibited by globomycin, a specific inhibitor of signal peptidase II. The gene region identified shows a high degree of homology to a gene region reported to be required for Pseudomonas aeruginosa type IV pilus production.     

Molecular cloning of the gene encoding an outer-membrane-associated beta-N-acetylglucosaminidase involved in chitin degradation system of Alteromonas sp. strain O-7

The gene encoding beta-N-acetylglucosaminidase (GlcNAcaseA) was cloned using PCR with degenerate oligonucleotide primers from the partial amino acid sequence of the enzyme. The gene encoded a polypeptide of 863 amino acids with a predicted molecular mass of 97kDa. A characteristic signal peptide, which was present at the amino-terminus of the precursor protein, contained four amino acids (Ala-Gly-Cys-Ser) identical in sequence and location to the processing and modification sites of the outer membrane lipoprotein of Escherichia coli, indicating that the mature GlcNAcaseA is a lipoprotein the N-terminal cysteine residue of which would be modified by the fatty acid that anchors the protein in the membrane. The predicted amino acid sequence of GlcNAcaseA showed similarity to bacterial beta-N-acetylglucosaminidases belonging to the family 20 glycosyl hydrolases.     

Nucleotide sequence of the iucD gene of the pColV-K30 aerobactin operon and topology of its product studied with phoA and lacZ gene fusions.

Gene iucD of the aerobactin operon of the Escherichia coli plasmid ColV-K30 encodes a membrane-bound enzyme synthesizing N6-hydroxylysine, the first product of the aerobactin biosynthesis pathway. The entire nucleotide sequence of the cloned iucD gene was determined, from which the primary and some aspects of the secondary structure of the encoded peptide were deduced. E. coli cells harboring multicopy plasmid pVLN12 (iucD+) hyperproduced an approximately 50-kilodalton peptide which was purified and identified as the product of the gene by examination of its amino-terminal sequence. Two iucD'-'lacZ gene fusions were constructed in vitro and four iucD'-'phoA gene fusions were generated in vivo by mutagenesis of iucD with transposon TnphoA (Tn5 IS50L::phoA). Analysis of the corresponding fusion proteins suggested at least two domains of attachment of the IucD protein to the inner side of the cytoplasmic membrane. The first apparent membrane-bound domain was found within the first 25 amino acids of the protein and showed a sequence which resembled that of the signal peptides.