Molecular characterization of the iron-hydroxamate uptake system in Staphylococcus aureus

To investigate iron uptake, a chromosomal locus containing three consecutive open reading frames, designated fhuC, fhuB, and fhuD, was identified in Staphylococcus aureus. Whereas the fhuC gene encodes an ATP-binding protein, fhuB and fhuD code for ferrichrome permeases and thus resemble an ATP-binding cassette transporter. A fhuB knockout mutant showed impaired uptake of iron bound to the siderophores but not of ferric chloride, suggesting that this operon is specific for siderophore-mediated iron uptake.     

Nucleotide sequence of the fhuC and fhuD genes involved in iron (III) hydroxamate transport: domains in FhuC homologous to ATP-binding proteins

Summary The transport of Fe³⁺ into cells of Escherichia coli occurs via siderophores and the uptake through the outer membrane of three Fe³⁺-siderophore compounds containing hydroxamate residues requires three specific receptor proteins. In contrast, transport through the cytoplasmic membrane is catalysed by three common proteins encoded by the fhuB, fhuC and fhuD genes. The nucleotide sequence of a DNA fragment containing the fhuC and fhuD genes has been determined: the open reading frame of fhuC contains 795 nucleotides which encode a polypeptide with a molecular weight of 29 255 and the largest open reading frame of the fhuD region comprises 888 nucleotides. However, we propose that translation of fhuD initiates at the fourth potential start codon resulting in a polypeptide with a molecular weight of 28 282. Both proteins are moderately nonpolar and membrane-bound. They lack obvious signal sequences. Segments of the FhuC protein display strong homology to ATP-binding proteins, suggesting a function in Fe³⁺ uptake similar to the ATP-binding proteins of transport systems that depend on periplasmic proteins. This study completes the nucleotide sequence of the fhu operon which consists of the four genes fhuA fhuC fhuD fhuB arranged in this order on the E. coli chromosome and transcribed from fhuA to fhuB.     

Albomycin uptake via a ferric hydroxamate transport system of Streptococcus pneumoniae R6

The antibiotic albomycin is highly effective against Streptococcus pneumoniae, with an MIC of 10 ng/ml. The reason for the high efficacy was studied by measuring the uptake of albomycin into S. pneumoniae. Albomycin was transported via the system that transports the ferric hydroxamates ferrichrome and ferrioxamine B. These two ferric hydroxamates antagonized the growth inhibition by albomycin and salmycin. Cross-inhibition of the structurally different ferric hydroxamates to both antibiotics can be explained by the similar iron coordination centers of the four compounds. [(55)Fe(3+)]ferrichrome and [(55)Fe(3+)]ferrioxamine B were taken up by the same transport system into S. pneumoniae. Mutants in the adjacent fhuD, fhuB, and fhuG genes were transport inactive and resistant to the antibiotics. Albomycin, ferrichrome, ferrioxamine B, and salmycin bound to the isolated FhuD protein and prevented degradation by proteinase K. The fhu locus consisting of the fhuD, fhuB, fhuG, and fhuC genes determines a predicted ABC transporter composed of the FhuD binding lipoprotein, the FhuB and FhuG transport proteins, and the FhuC ATPase. It is concluded that active transport of albomycin mediates the high antibiotic efficacy in S. pneumoniae.     

Transport of ferric-aerobactin into the periplasm and cytoplasm of Escherichia coli K12: role of envelope-associated proteins and effect of endogenous siderophores.

Purified [14C]aerobactin, supplied exogenously to non-growing bacteria, was translocated via the periplasm into the cytoplasm of Escherichia coli K12 strains expressing the aerobactin receptor protein IutA. No significant uptake was observed into either compartment of strains lacking the iutA gene or specifically defective in tonB. Uptake into both compartments was markedly reduced, but not abolished, in an exb mutant. Accumulation of [14C]aerobactin in the periplasm of fhuD, fhuB or fhuC mutant strains was not significantly lower than in the wild-type strain, but entry into the cytoplasm was greatly reduced in all cases. Uptake of aerobactin by strains wild-type for all transport functions occurred most efficiently in strains either lacking or specifically defective in the genetic determinants for aerobactin biosynthesis; significantly lower levels of exogenous 14C-labelled siderophore were observed in both compartments of strains producing aerobactin. Aerobactin-mediated 59Fe uptake, however, was not inhibited by the presence of endogenous aerobactin. Endogenous enterochelin did not affect aerobactin uptake.     

Functions in outer and inner membranes of Escherichia coli for ferrichrome transport.

Mutants of the fhuA gene of Escherichia coli K-12, which encodes a receptor protein in the outer membrane, took up ferrichrome after exposure to pronase, whereas fhuB mutants remained transport negative. The latter finding supports our previous proposal that fhuB mutants are defective in a function that residues in the cytoplasmic membrane. Cells remained completely viable after treatment with pronase, although they became sensitive to the antibiotic actinomycin.     

Ferrichrome utilization in a mesorhizobial population: microevolution of a three-locus system

The ability to utilize the siderophore ferrichrome as an iron source was found to be a variable trait in a field population of mesorhizobia. To investigate the genetic basis of this variation, genes required for ferrichrome utilization (fhu genes) were characterized in Mesorhizobium strain R88B, an Fhu(+) member of the population. Functional fhu genes were present at three loci. Two genes of the ferrichrome ABC transporter, fhuBD, were identified at an fhu1 locus downstream of the symbiosis island that was integrated at the phe-tRNA gene. The fhuA gene encoding the ferrichrome outer membrane receptor was located in the fhu2 locus together with non-functional fhuDB genes, while the fhuC gene encoding the ATPase required for ferrichrome transport was part of the fhu3 locus that included genes required to form a functional TonB complex. None of the fhu genes were present in the sequenced Mesorhizobium loti strain MAFF303099. Comparisons with MAFF303099 suggested that the fhu2 and fhu3 loci evolved through small-scale (< 5 kb) acquisitions and deletions. Despite their independent origins, the three fhu loci were coordinately regulated in response to iron availability. Within the mesorhizobial population, the ability to utilize ferrichrome was most strongly correlated with the presence of the fhuA gene. We hypothesize that the ferrichrome transport system evolved through cycles of gene acquisition and deletion, with the positive selection pressure of an iron-poor or siderophore-rich environment being offset by the negative pressure of the outer membrane receptor being a target for phage.     

Chromosomal genes for ColV plasmid-determined iron(III)-aerobactin transport in Escherichia coli.

Four chromosomal genes, tonA (fhuA), fhuB, tonB, and exbB, were required for the transport of iron(III)-aerobactin specified by the plasmids ColV-K311, ColV-K229, ColV-K328, and ColV-K30. These genes also determine the transport system in Escherichia coli for the iron ionophore ferrichrome. Aerobactin and ferrichrome are both iron ligands of the hydroxamate type, but they are of different structure. The ColV plasmids determine an outer membrane protein that serves as a receptor for cloacin. Cloacin-resistant mutants were devoid of iron(III)-aerobactin transport but were unimpaired in ferrichrome transport. We conclude that for iron(III)-aerobactin transport two outer membrane proteins, the TonA and the cloacin receptor protein, have to interact functionally or structurally or both.     

fhuC and fhuD genes for iron (III)-ferrichrome transport into Escherichia coli K-12.

The nucleotide sequence for a 1,900-base-pair region of the Escherichia coli chromosome that includes the genes fhuC and fhuD was determined. Within this sequence are two open reading frames: nucleotides 127 to 921 and nucleotides 924 to 1811. These coding regions specify a FhuC protein with an Mr of 28,423 and a mature FhuD protein with an Mr of 29,610. The deduced amino acid sequence of FhuC shows extensive homology with those of components of some bacterial transport systems which are peripheral proteins of the cytoplasmic membrane. Because the FhuD protein contains a typical signal sequence of 30 amino acids at the amino terminus and displays characteristics of a soluble protein, it may be exported into the periplasm.     

Iron-hydroxamate transport into Escherichia coli K12: localization of FhuD in the periplasm and of FhuB in the cytoplasmic membrane

Summary ThefhuB, fhuC andfhuD genes encode proteins which catalyze transport of iron(III)-hydroxamate compounds from the periplasm into the cytoplasm ofEscherichia coli. ThefhuB, C, D genes were cloned downstream of a strong phage T7 promoter and transcribed by T7 RNA polymerase. The overexpressed FhuD protein appeared in two forms of 31 and 28 kDa and was released upon conversion of vegetative cells into spheroplasts, suggesting synthesis of FhuD as a precursor and export into the periplasm. The very hydrophobic FhuB protein was found in the cytoplasmic membrane. These properties, together with the previously found homologies in the FhuC protein to ATP-binding proteins, display the characteristics of a periplasmic binding protein dependent transport system across the cytoplasmic membrane. The molecular weight of FhuB and the sequence offhuC, as previously published by us, was confirmed. FhuB exhibited double the size of most hydrophobic proteins of such systems and showed homology between the amino- and carboxy-terminal halves of the protein, indicating duplication of an original gene and subsequent fusion of the two DNA fragments.     

Involvement of ExbB and TonB in transport across the outer membrane of Escherichia coli: phenotypic complementation of exb mutants by overexpressed tonB and physical stabilization of TonB by ExbB.

The exb locus in Escherichia coli consists of two genes, termed exbB and exbD. Exb functions are related to TonB function in that most TonB-dependent processes are enhanced by Exb. Like tonB mutants, exb mutants were resistant to colicin M and albomycin but, in contrast to tonB mutants, showed only reduced sensitivity to colicins B and D. Overexpressed tonB on the multicopy vector pACYC177 largely restored the sensitivity of exb mutants to colicins B, D, and M but only marginally increased sensitivity to albomycin. Suppression of the btuB451 mutation in the structural gene for the vitamin B12 outer membrane receptor protein by a mutation in tonB occurred only in an exb+ strain. Degradation of the unstable overproduced TonB protein was prevented by overproduced ExbB protein. The ExbB protein also stabilized the ExbD protein. Pulse-chase experiments with radiolabeled ferrichrome revealed release of ferrichrome from exbB, tonB, and fhuC mutants, showing that ferrichrome had not crossed the cytoplasmic membrane. It is concluded that the ExbB and ExbD proteins contribute to the activity of TonB and, like TonB, are involved in receptor-dependent transport processes across the outer membrane.