Organization of genes encoding membrane proteins of the Escherichia coli ferrienterobactin permease

Transposon mutagenesis and plasmid complementation studies have identified two genes, fepD and fepG, which are essential for ferrienterobactin transport in Escherichia coli. These genes mapped in the enterobactin gene cluster and genetic evidence indicated that they are transcribed as part of an operon (fepD, fepG, fepC). The nucleotide sequence of fepD was determine; it could encode a hydrophobic 33.8 kDa protein with sequence homologies to other iron and vitamin B12 transport proteins. Also identified, between fepD and fepB, was an open reading frame (ORF43) with no detectable function; its 43 kDa protein product (P43) was seen on polyacrylamide gels. The fepD-C operon and ORF43 were divergently transcribed from a 110bp region containing a binding site for the repressor protein Fur.     

Escherichia coli K-12 envelope proteins specifically required for ferrienterobactin uptake.

Escherichia coli genes specifically required for transport of iron by the siderophore enterobactin are designated fep. The studies reported here were initiated to identify and localize the fepB product. The plasmid pCP111, which consisted of an 11-kilobase E. coli DNA fragment containing fepB ligated to pACYC184, was constructed. The fepB gene was subcloned; in the process, complementation tests and Tn5 mutagenesis results provided evidence for the existence of a new fep gene, fepC. The order of the transport genes in the ent gene cluster is as follows: fepA fes entF fepC fepB entE. Minicell, maxicell, and in vitro DNA-directed protein synthesizing systems were used to identify the fepB and fepC products. The fepC polypeptide was 30,500 daltons in standard sodium dodecyl sulfate-polyacrylamide gels. The fepB gene was responsible for the appearance of three or four bands (their apparent molecular weights ranged from 31,500 to 36,500) in sodium dodecyl sulfate-polyacrylamide gels, depending on the gel system employed. The largest of these was tentatively designated proFepB, since it apparently had a leader sequence. Localization experiments showed that FepC was a membrane constituent and that mature FepB was present in the periplasm. An additional polypeptide (X) was also encoded by the bacterial DNA of pCP111, but its relationship to iron transport is unknown. The results indicated that ferrienterobactin uptake is mediated by a periplasmic transport system and that genes coding for outer membrane (fepA), periplasmic (fepB), and cytoplasmic membrane (fepC) components have now been identified.     

A multifunctional ATP-binding cassette transporter system from Vibrio cholerae transports vibriobactin and enterobactin

Vibrio cholerae uses the catechol siderophore vibriobactin for iron transport under iron-limiting conditions. We have identified genes for vibriobactin transport and mapped them within the vibriobactin biosynthetic gene cluster. Within this genetic region we have identified four genes, viuP, viuD, viuG and viuC, whose protein products have homology to the periplasmic binding protein, the two integral cytoplasmic membrane proteins, and the ATPase component, respectively, of other iron transport systems. The amino-terminal region of ViuP has homology to a lipoprotein signal sequence, and ViuP could be labeled with [(3)H]palmitic acid. This suggests that ViuP is a membrane lipoprotein. The ViuPDGC system transports both vibriobactin and enterobactin in Escherichia coli. In the same assay, the E. coli enterobactin transport system, FepBDGC, allowed the utilization of enterobactin but not vibriobactin. Although the entire viuPDGC system could complement mutations in fepB, fepD, fepG, or fepC, only viuC was able to independently complement the corresponding fep mutation. This indicates that these proteins usually function as a complex. V. cholerae strains carrying a mutation in viuP or in viuG were constructed by marker exchange. These mutations reduced, but did not completely eliminate, vibriobactin utilization. This suggests that V. cholerae contains genes in addition to viuPDGC that function in the transport of catechol siderophores.     

Membrane disposition of the Escherichia coli mannitol permease: identification of membrane-bound and cytoplasmic domains

Two proteolytic fragments of the Escherichia coli mannitol permease (EIImtl) have been identified on autoradiograms of sodium dodecyl sulfate-polyacrylamide gels and mapped with respect to the membrane. EIImtl was selectively radiolabeled with either [35S]methionine or a mixture of 14C-labeled amino acids in E. coli minicells harboring a plasmid containing the mannitol operon. The intact permease (Mr 65,000) in everted vesicles derived from labeled minicells was cleaved by mild trypsinolysis into two smaller fragments (Mr 34,000 and 29,000). The 34,000-dalton fragment remained in the membrane and was insensitive to further proteolysis by trypsin. This fragment was identified as the N-terminal half of the protein by comparing the amount of the original [35S]methionine label that it retained with the known differential distribution of methionine in the two halves of EIImtl. The 29,000-dalton fragment, which was released into the soluble fraction and was sensitive to further trypsinolysis, therefore corresponds to the C-terminal half of the mannitol permease. Both fragments were shown to be antigenically related to EIImtl by immunoblotting with anti-EIImtl antibody. The 34,000-dalton fragment was further shown to form an oligomer under conditions which allow the intact enzyme to dimerize, suggesting that this domain plays an important role in EIImtl subunit interactions. These results support a model in which EIImtl consists of two domains of approximately equal size: a membrane-bound, N-terminal domain with a tendency to self-associate, and a cytoplasmic C-terminal domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular analysis of the Escherichia coli ferric enterobactin receptor FepA

In Escherichia coli, the outer membrane protein FepA is a receptor for the siderophore complex ferric enterobactin and for colicins B and D. To identify protein domains important for FepA activity, the effects of deletion and linker insertion mutations on receptor structure and function were examined. In-frame internal deletion mutations removing sequences encoding up to 304 amino acid residues resulted in functionally defective FepA polypeptides, although most were translocated efficiently to the outer membrane. One exception, a derivative lacking 87 internal amino acid residues near the N terminus, showed an inability to transport ferric enterobactin but retained limited colicin receptor function. Analysis of cells carrying 3'-terminal fepA deletion mutations suggested that residues within the C terminus of FepA may be involved in secretion and proper translocation of the protein to the outer membrane. Introduction of the peptide Leu-Glu after FepA residues 55, 142, or 324 severely impaired receptor function for all three ligands, while the same insertion after residues 339 or 359 had virtually no detrimental effect on FepA function. Foreign peptides inserted after residues 204 or 635 restricted colicin B and D function only, leaving ferric enterobactin transport ability at near wild-type levels. The results presented in this study have identified key regions of FepA potentially involved in receptor function and demonstrate the presence of both shared and unique ligand-responsive domains.     

Haem iron-transport system in enterohaemorrhagic Escherichia coli O157:H7

In this study, we identified the iron-transport systems of Escherichia coli O157:H7 strain EDL933. This strain synthesized and transported enterobactin and had a ferric citrate transport system but lacked the ability to produce or use aerobactin. It used haem and haemoglobin, but not transferrin or lactoferrin, as iron sources. We cloned the gene encoding an iron-regulated haem-transport protein and showed that this E. coli haem-utilization gene ( chuA ) encoded a 69 kDa outer membrane protein that was synthesized in response to iron limitation. Expression of this protein in a laboratory strain of E. coli was sufficient for utilization of haem or haemoglobin as iron sources. Mutation of the chromosomal chuA and tonB genes in E. coli O157:H7 demonstrated that the utilization of haemin and haemoglobin was ChuA- and TonB-dependent. Nucleotide sequence analysis of chuA revealed features characteristic of TonB-dependentFur-regulated, outer membrane iron-transport proteins. It was highly homologous to the shuA gene of Shigella dysenteriae and less closely related to hemR of Yersinia enterocolitica and hmuR of Yersinia pestis . A conserved Fur box was identified upstream of the chuA gene, and regulation by Fur was confirmed.     

Involvement of inner and outer membrane components in the transport of iron and in colicin B action in Escherichia coli.

Spheroplasts of Escherichia coli mutants were used to investigate the roles of the inner and outer membranes in the transport of iron. tonA mutants, known to be defective in an outer membrane component of the ferrichrome transport system, regained the ability to transport ferrichrome when converted to spheroplasts. On the other hand, the tonB mutant was unable to transport ferric enterochelin in either whole cells or spheroplasts. This implies that an element of the inner membrane is affected. fep mutants were also unable to transport ferric enterochelin, and fell into two classes, fepA and fepB. Spheroplasts of the former class transported ferric enterochelin, and those of the latter did not. This implies that the fepA mutants are defective in ferric enterochelin transport across the outer membrane, and that fepB mutants probably lack the facility to transport ferric enterochelin across the inner membrane. Colicin B action on fepA mutants was found to differ from that on fepB mutants.     

Export of the siderophore enterobactin in Escherichia coli: involvement of a 43 kDa membrane exporter

The enterobactin system for iron transport in Escherichia coli is well characterized with the exception of the mechanism of enterobactin secretion to the extracellular environment. Escherichia coli membrane protein P43, encoded by ybdA in the chromosomal region of genes involved in enterobactin synthesis, shows strong homology to the 12-transmembrane segment major facilitator superfamily of export pumps. A P43-null mutation was created and siderophore nutrition assays, performed with a panel of E. coli strains expressing one or more outer membrane receptors for enterobactin-related compounds, demonstrated that the P43 mutant was unable to secrete enterobactin efficiently. Products released from the mutant strain were identified with thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), revealing that the P43 mutant secretes little, if any, enterobactin, but elevated levels of enterobactin breakdown products 2,3- dihydroxybenzoylserine (DHBS) monomer, dimer, and trimer. These data establish that P43 is a critical component of the E. coli enterobactin secretion machinery and provides a rationale for the designation of the previous genetic locus ybdA as entS to reflect its relevant biological function.     

Recognition and transport of ferric enterobactin in Escherichia coli.

The specificity of the outer membrane protein receptor for ferric enterobactin transport in Escherichia coli and the mechanism of enterobactin-mediated transport of ferric ions across the outer membrane have been studied. Transport kinetic and inhibition studies with ferric enterobactin and synthetic structural analogs have mapped the parts of the molecule important for receptor binding. The ferric complex of the synthetic structural analog of enterobactin, 1,3,5-N,N',N'-tris-(2,3-dihydroxybenzoyl)triaminomethylbenzene (MECAM), was transported with the same maximum velocity as was ferric enterobactin. A double-label transport assay with [59Fe, 3H]MECAM showed that the ligand and the metal are transported across the outer membrane at an identical rate. Under the growth conditions used, large fractions of the transported complexes were available for exchange across the outer membrane when a large excess of extracellular complex was added to the cell suspension; at least 60% of the internalized [59Fe]enterobactin exchanged with extracellular [55Fe]enterobactin. Internalized [59Fe, 3H]MECAM was released from the cell as the intact complex when either unlabeled Fe-MECAM or Fe-enterobactin was added extracellularly. The results suggest a mechanism of active transport of unmodified coordination complex across the outer membrane with possible accumulation in the periplasm.     

Ferric enterobactin binding and utilization by Neisseria gonorrhoeae

FetA, formerly designated FrpB, an iron-regulated, 76-kDa neisserial outer membrane protein, shows sequence homology to the TonB-dependent family of receptors that transport iron into gram-negative bacteria. Although FetA is commonly expressed by most neisserial strains and is a potential vaccine candidate for both Neisseria gonorrhoeae and Neisseria meningitidis, its function in cell physiology was previously undefined. We now report that FetA functions as an enterobactin receptor. N. gonorrhoeae FA1090 utilized ferric enterobactin as the sole iron source when supplied with ferric enterobactin at approximately 10 microM, but growth stimulation was abolished when an omega (Omega) cassette was inserted within fetA or when tonB was insertionally interrupted. FA1090 FetA specifically bound 59Fe-enterobactin, with a Kd of approximately 5 microM. Monoclonal antibodies raised against the Escherichia coli enterobactin receptor, FepA, recognized FetA in Western blots, and amino acid sequence comparisons revealed that residues previously implicated in ferric enterobactin binding by FepA were partially conserved in FetA. An open reading frame downstream of fetA, designated fetB, predicted a protein with sequence similarity to the family of periplasmic binding proteins necessary for transporting siderophores through the periplasmic space of gram-negative bacteria. An Omega insertion within fetB abolished ferric enterobactin utilization without causing a loss of ferric enterobactin binding. These data show that FetA is a functional homolog of FepA that binds ferric enterobactin and may be part of a system responsible for transporting the siderophore into the cell.     

Molecular characterization of the iron transport system mediated by the pJM1 plasmid in Vibrio anguillarum 775.

Complementation of insertion mutants showed that the polypeptides FatD, FatC, FatB, and FatA are essential for the iron-transport process encoded by pJM1. Sequence analysis followed by homology studies indicated that transport of ferric anguibactin into Vibrio anguillarum 775 follows the same mechanism as reported for transport of Fe(3+)-hydroxamates, Fe(3+)-catecholates, ferric dicitrate, and vitamin B12 into Escherichia coli. Homology of FatA, part of the receptor complex, to seven E. coli receptor proteins involved in uptake of siderophores and vitamin B12 supports the idea of a common ancestral gene. A "TonB-Box" was found in FatA suggesting the existence of a TonB-like protein function in V. anguillarum. A high homology in the primary structure of FatB to FhuD, FecB, FepB, and BtuE suggests that FatB is the anguibactin-binding protein located in the periplasmic space. FatD and FatC are polytopic integral membrane proteins. According to their homologies to other proteins from other transport systems, they may be involved in the translocation of ferric anguibactin across the cytoplasmic membrane.     

Binding of ferric enterobactin by the Escherichia coli periplasmic protein FepB

The periplasmic protein FepB of Escherichia coli is a component of the ferric enterobactin transport system. We overexpressed and purified the binding protein 23-fold from periplasmic extracts by ammonium sulfate precipitation and chromatographic methods, with a yield of 20%, to a final specific activity of 15,500 pmol of ferric enterobactin bound/mg. Periplasmic fluid from cells overexpressing the binding protein adsorbed catecholate ferric siderophores with high affinity: in a gel filtration chromatography assay the K(d) of the ferric enterobactin-FepB binding reaction was approximately 135 nM. Intrinsic fluorescence measurements of binding by the purified protein, which were more accurate, showed higher affinity for both ferric enterobactin (K(d) = 30 nM) and ferric enantioenterobactin (K(d) = 15 nM), the left-handed stereoisomer of the natural E. coli siderophore. Purified FepB also adsorbed the apo-siderophore, enterobactin, with comparable affinity (K(d) = 60 nM) but did not bind ferric agrobactin. Polyclonal rabbit antisera and mouse monoclonal antibodies raised against nearly homogeneous preparations of FepB specifically recognized it in solid-phase immunoassays. These sera enabled the measurement of the FepB concentration in vivo when expressed from the chromosome (4,000 copies/cell) or from multicopy plasmids (>100,000 copies/cell). Overexpression of the binding protein did not enhance the overall affinity or rate of ferric enterobactin transport, supporting the conclusion that the rate-limiting step of ferric siderophore uptake through the cell envelope is passage through the outer membrane.     

Identification of hydrophobic proteins FepD and FepG of the Escherichia coli ferrienterobactin permease.

In Escherichia coli, iron assimilation by means of the siderophore enterobactin requires two hydrophobic cytoplasmic membrane proteins, FepD and FepG, which are essential components of a binding-protein-dependent transport system. Such components are typically difficult to detect. Here we report observation of the fepD and fepG gene products in polyacrylamide gels; they appeared as diffuse bands at positions consistent with smaller sizes than those predicted by sequence analysis. Translational coupling was suggested by the lack of a detectable product from the fepG message in the absence of translation of the upstream fepD message. The orientation of FepD/FepG in the membrane was predicted based on their similarities in sequence and hydrophobicity to FhuB.     

Transport of hemolysin by Escherichia coli

The hemolytic phenotype in Escherichia coli is determined by four genes. Two (hlyC and hlyA) determine the synthesis of a hemolytically active protein which is transported across the cytoplasmic membrane. The other two genes (hlyBa and hlyBb) encode two proteins which are located in the outer membrane and seem to form a specific transport system for hemolysin across the outer membrane. The primary product of gene hlyA is a protein (protein A) of 106,000 daltons which is nonhemolytic and which is not transported. No signal peptide can be recognized at its N-terminus. In the presence of the hlyC gene product (protein C), the 106,000-dalton protein is processed to the major proteolytic product of 58,000 daltons, which is hemolytically active and is transported across the cytoplasmic membrane. Several other proteolytic fragments of the 106,000-dalton protein are also generated. During the transport of the 58,000-dalton fragment (and possible other proteolytic fragments of hlyA gene product), the C protein remains in the cytoplasm. In the absence of hlyBa and hlyBb the entire hemolytic activity (mainly associated with the 58,000-dalton protein) is located in the periplasm: Studies on the location of hemolysin in hlyBa and hlyBb mutants suggest that the gene product of hlyBa (protein Ba) binds hemolysin and leads it through the outer membrane whereas the gene product of hlyBb (protein Bb) releases hemolysin from the outer membrane. This transport system is specific for E coli hemolysin. Other periplasmic enzymes of E coli and heterologous hemolysin (cereolysin) are not transported.