Characterization of a chromosomal mutant that blocks hemolysin excretion in Escherichia coli

Abstract Alanine dipeptides normally penetrate into Pseudomonas aeruginosa by the way of two transport systems. In peptidase N-deficient mutants, dialanine is unable to use its low affinity transport system. Uptake competition showed that this system harboured a permease common to the transport of the amino acid alanine. This permease permits the penetration of both alanine and alanyl peptides uniquely in the presence of active peptidase N. The uptake of trialanine is independent of the presence of active peptidase N inside bacteria despite the fact that hydrolysis of this tripeptide absolutely requires this activity.     

Uptake and conversion of the antibiotic albomycin by Escherichia coli K-12.

The antibiotic albomycin is transported into cells of Escherichia coli K-12 by the same uptake system as the iron-supplying ferrichrome complex. The iron-complexing hydroxamate moieties of albomycin and ferrichrome are structurally similar. During the phase of rapid iron uptake the chelators were not found in the cells. In order to understand the antibiotic activity of albomycin, it was labeled in the hydroxamate with tritium and in the presumed antibiotically active area with radioactive sulfur. While the tritium label was not retained by the cells, part of the sulfur label was taken up and concentrated 500-fold within the cell. The sulfur was not incorporated into proteins or nucleic acids since it was recovered as a low molecular weight component. Gel filtration on Bio-Gel P-2 revealed one tritium-labeled and two sulfur-labeled cleavage products in the incubation medium. We conclude that albomycin is actively transported via its ferrichrome-like portion into the cells and that the growth-inhibitory moiety is released by hydrolysis intracellularly and remains there.     

Iron(III) hydroxamate transport of Escherichia coli: restoration of iron supply by coexpression of the N- and C-terminal halves of the cytoplasmic membrane protein FhuB cloned on separate plasmids

Summary Transport of iron(III) hydroxamates across the inner membrane into the cytoplasm of Escherichia coli cells is mediated by the FhuC, FhuD and FhuB proteins. We studied the extremely hydrophobic FhuB protein (70 kDa) which is located in the cytoplasmic membrane. The N- and C-terminal halves of the protein [FhuB(N) and FhuB(C)] show homology to each other and to the equivalent polypeptides involved in uptake of ferric dicitrate and of vitamin B₂. Various plasmids carrying only one-half of the fhuB gene were expressed in fhuB ^– mutants. Only combinations of FhuB(N) and FhuB(C) polypeptides restored sensitivity to albomycin and growth on iron hydroxamates as sole iron source; no activity was obtained with either half of FhuB alone. These results indicate that both halves of FhuB are essential for substrate translocation and that they combine to form an active permease when expressed separately. In addition, a FhuB derivative with a large internal duplication of 271 amino acids was found to be partially active in transport, indicating that the extra portion did not perurb proper insertion of the active FhuB segments into the cytoplasmic membrane.     

Iron uptake in Salmonella typhimurium: utilization of exogenous siderochromes as iron carriers

Aerobic microorganisms have evolved a variety of siderochromes, special ligands which can dissolve insoluble ferric iron and facilitate its transport into the cell. We have found that enb mutants of Salmonella typhimurium blocked in the biosynthesis of enterobactin (its natural iron carrier) are able to utilize siderochromes of different types made by other microorganisms as iron carriers. The antibiotic albomycin delta(2) was used to select mutants defective in ferrichrome-mediated iron uptake. Twelve classes of albomycin-resistant mutants, named sid, were defined on the basis of their growth responses to other siderochromes. Most of these classes have genetic lesions in loci that are cotransduced with panC (represented at 9 min on the genetic map). The locus designated sidJ is cotransduced with enb, whereas sidK and sidL are linked with neither panC nor enb. Genetic and physiological data indicate that S. typhimurium has several transport systems of high specificity for a variety of siderochromes produced by other microorganisms.     

Biosynthesis of Albomycin δ(2) Provides a Template for Assembling Siderophore and Aminoacyl-tRNA Synthetase Inhibitor Conjugates

"Trojan horse" antibiotic albomycins are peptidyl nucleosides consisting of a highly modified 4'-thiofuranosyl cytosine moiety and a ferrichrome siderophore that are linked by a peptide bond via a serine residue. While the latter component serves to sequester iron from the environment, the seryl nucleoside portion is a potent inhibitor of bacterial seryl-tRNA synthetases, resulting in broad-spectrum antimicrobial activities of albomycin δ(2). The isolation of albomycins has revealed this biological activity is optimized only following two unusual cytosine modifications, N4-carbamoylation and N3-methylation. We identified a genetic locus (named abm) for albomycin production in Streptomyces sp. ATCC 700974. Gene deletion and complementation experiments along with bioinformatic analysis suggested 18 genes are responsible for albomycin biosynthesis and resistance, allowing us to propose a potential biosynthetic pathway for installing the novel chemical features. The gene abmI, encoding a putative methyltransferase, was functionally assigned in vitro and shown to modify the N3 of a variety of cytosine-containing nucleosides and antibiotics such as blasticidin S. Furthermore, a ΔabmI mutant was shown to produce the descarbamoyl-desmethyl albomycin analogue, supporting that the N3-methylation occurs before the N4-carbamoylation in the biosynthesis of albomycin δ(2). The combined genetic information was utilized to identify an abm-related locus (named ctj) from the draft genome of Streptomyces sp. C. Cross-complementation experiments and in vitro studies with CtjF, the AbmI homologue, suggest the production of a similar 4'-thiofuranosyl cytosine in this organism. In total, the genetic and biochemical data provide a biosynthetic template for assembling siderophore-inhibitor conjugates and modifying the albomycin scaffold to generate new derivatives.     

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.     

Sideromycins: tools and antibiotics

Sideromycins are antibiotics covalently linked to siderophores. They are actively transported into gram-positive and gram-negative bacteria. Energy-coupled transport across the outer membrane and the cytoplasmic membrane strongly increases their antibiotic efficiency; their minimal inhibitory concentration is at least 100-fold lower than that of antibiotics that enter cells by diffusion. This is particularly relevant for gram-negative bacteria because the outer membrane, which usually forms a permeability barrier, in this case actively contributes to the uptake of sideromycins. Sideromycin-resistant mutants can be used to identify siderophore transport systems since the mutations are usually in transport genes. Two sideromycins, albomycin and salmycin, are discussed here. Albomycin, a derivative of ferrichrome with a bound thioribosyl-pyrimidine moiety, inhibts seryl-t-RNA synthetase. Salmycin, a ferrioxamine derivative with a bound aminodisaccharide, presumably inhibts protein synthesis. Crystal structures of albomycin bound to the outer membrane transporter FhuA and the periplasmic binding protein FhuD have been determined. Albomycin and salmycin have been used to characterize the transport systems of Escherichia coli and Streptococcus pneumoniae and of Staphylococcus aureus, respectively. The in vivo efficacy of albomycin and salmycin has been examined in a mouse model using Yersinia enterocolitica, S. pneumoniae, and S. aureus infections. Albomycin is effective in clearing infections, whereas salmycin is too unstable to lead to a large reduction in bacterial numbers. The recovery rate of albomycin-resistant mutants is lower than that of the wild-type, which suggests a reduced fitness of the mutants. Albomycin could be a useful antibiotic provided sufficient quantities can be isolated from streptomycetes or synthesized chemically.     

Genetics and regulation of peptidase N in Escherichia coli K-12

Escherichia coli K-12 strains contain a cytoplasmic activity, peptidase N, capable of hydrolyzing alanine-p-nitroanilide. Mutations in the structural gene for the enzyme, pepN, were mapped, and the properties of mutant strains were examined. The pepN locus lay between ompF and asnS at approximately 20.8 min on the E. coli chromosome. Loss of peptidase N activity through mutation had no apparent effect on the growth rate or nutritional needs of the cell. Enzyme levels in wild-type strains were constant throughout the growth cycle and were constitutive in all of the growth media tested. Starvation for carbon, nitrogen, or phosphate also did not alter enzyme levels. Constitutive expression of peptidase N is consistent with the idea that the enzyme plays a significant role in the degradation of intracellularly generated peptides.     

Iron(III) hydroxamate transport across the cytoplasmic membrane ofEscherichia coli

Summary Transport of iron(III) hydroxamates across the inner membrane into the cytoplasm ofEscherichia coli is mediated by the FhuC, FhuD and FhuB proteins and displays characteristics typical of a periplasmic-binding-protein-dependent transport mechanism. In contrast to the highly specific receptor proteins in the outer membrane, at least six different siderophores of the hydroxamate type and the antibiotic albomycin are accepted as substrates. AfhuB mutant (deficient in transport of substrates across the inner membrane) which overproduced the periplasmic FhuD 30-kDa protein, bound [⁵⁵Fe] iron(III) ferrichrome. Resistance of FhuD to proteinase K in the presence of ferrichrome, aerobactin, and coprogen indicated binding of these substrates to FhuD. FhuD displays significant similarity to the periplasmic FecB, FepB, and BtuE proteins. The extremely hydrophobic FhuB 70-kDa protein is located in the cytoplasmic membrane and consists of two apparently duplicated halves. The N-and C-terminal halves [FhuB(N) and FhuB(C)] were expressed separately infhuB mutants. Only combinations of FhuB(N) and FhuB(C) polypeptides restored sensitivity to albomycin and growth on iron hydroxamate as a sole iron source, indicating that both halves of FhuB were essential for substrate translocation and that they combined to form an active permease. In addition, a FhuB derivative with a large internal duplication of 271 amino acids was found to be transport-active, indicating that the extra portion did not disturb proper insertion of the active FhuB segments into the cytoplasmic membrane. A region of considerable similarity, present twice in FhuB, was identified near the C-terminus of 20 analyzed hydrophobic proteins of periplasmic-binding-protein-dependent systems. The FhuC 30 kDa protein, most likely involved in ATP binding, contains two domains representing consensus sequences among all peripheral cytoplasmic membrane proteins of these systems. Amino acid replacements in domain I (LysGlu and Gln) and domain II (AspAsn and Glu) resulted in a transport-deficient phenotype.     

Lantibiotic transporter requires cooperative functioning of the peptidase domain and the ATP binding domain

Lantibiotics are ribosomally synthesized and post-translationally modified peptide antibiotics that contain unusual amino acids such as dehydro and lanthionine residues. Nukacin ISK-1 is a class II lantibiotic, whose precursor peptide (NukA) is modified by NukM to form modified NukA. ATP-binding cassette (ABC) transporter NukT is predicted to cleave off the N-terminal leader peptide of modified NukA and secrete the mature peptide. Multiple sequence alignments revealed that NukT has an N-terminal peptidase domain (PEP) and a C-terminal ATP binding domain (ABD). Previously, in vitro reconstitution of NukT has revealed that NukT peptidase activity depends on ATP hydrolysis. Here, we constructed a series of NukT mutants and investigated their transport activity in vivo and peptidase activity in vitro. Most of the mutations of the conserved residues of PEP or ABD resulted in failure of nukacin ISK-1 production and accumulation of modified NukA inside the cells. NukT(N106D) was found to be the only mutant capable of producing nukacin ISK-1. Asn(106) is conserved as Asp in other related ABC transporters. Additionally, an in vitro peptidase assay of NukT mutants demonstrated that PEP is on the cytosolic side and all of the ABD mutants as well as PEP (with the exception of NukT(N106D)) did not have peptidase activity in vitro. Taken together, these observations suggest that the leader peptide is cleaved off inside the cells before peptide secretion; both PEP and ABD are important for NukT peptidase activity, and cooperation between these two domains inside the cells is indispensable for proper functioning of NukT.     

Fuctional organization of the outer membrane of escherichia coli: Phage and colicin receptors as components of iron uptake systems

The functional interaction of outer memberane proteins of E. coli can be studied using phage and colicin receptors which are essential components of penetration systems. The uptake of ferric iron in the form of the ferrichrome complex requires the ton A and ton B functions in the outer membrane of E. coli. The ton A gene product is the receptor protein for phage T5 and is required together with the ton B function by the phages T1 anf ?80 to infect cells and by colicin M and the antibiotic albomycin, a structural analogue of ferrichrome, to kill cells. The ton B function is necessary for the uptake of ferric iron complexed by citrate. Iron complexed by enterochelin is only transported in the presence of the ton B and feu functions. Cells which have lost the feu function are resistant to the colicins B, I or V while ton B mutants are resistant to all colicins. The interaction of the ton A, Ton B, and feu functions apparently permits quite different “substrates” to overcome the permeablility barrier of the outer membrane. It was shown for ferrichrome dependent iron uptake that the complexing agent was not altered and could be used repeatedly. Only very low amounts of ³H-labeled ferrichrome were found in the cell. It is possible that the iron is mobilized in the membrane and that desferriferrichrome is released into the medium without having entered the cytoplasm. Growth on ferrichrome as the sole iron source waw used to select revertants of T5 resistant ton A mutants. All revertants exhibited wild-type properties with the exception of partial revertants. In these 4 strains, as in the ton A mutants, the ton A protein was not detectable by SDS polyacrylamide gel electrophoreses of outer membranes. Albomycin resistant mutants were selected and shown to fall into 5 categories: (1) ton A; (2) ton B mutants; (3) mutants with no iron transport defects and normal ton A/ton B functions, which might be target site mutants; (4) mutants which were deficient in ferrichrome-mediated iron uptake but had normal ton A/ton B functions. We tentatively consider that the defect might be located in the active transport system of the cytoplasmic membrane; (5) a variety of mutants with the following general properties: most of them were resistant to colicin M, transported iron poorly, and, like ton B mutants, contained additional proteins in the outer membrane. The outer membrane protein patterns of wild-type and ton B mutant strains were compared by slab gel electrophoresis in an attempt to identify a ton B protein. It was observed that under most growth conditions, ton B mutants overproduced 3 proteins of molecular weights 74,000–83,000. In extracted, iron-deficient medium, both the wild-type and ton B mutant strains had similar large amounts of these proteins in their outer membranes. The appearance of these proteins was suppressed by excess iron in both wild-type and mutant. From this evidence it is apparent that the proteins appear as a response to low intracellular iron rather than being controlled by the ton B gene. The nature of these proteins and their possible role in iron transport is disussed.     

Anterograde axonal transport of Boc-Arg-Val-Arg-Arg-MCA hydrolyzing enzyme in rat sciatic nerves: cleavage occurs between basic residues

Axonal transport of Boc-Arg-Val-Arg-Arg-MCA hydrolyzing enzyme activity was studied in rat sciatic nerves from 12 to 120 h after double ligations. The anterograde axonal transport increased and peaked 72 h after ligation. The optimum pH for Boc-Arg-Val-Arg-Arg-MCA hydrolyzing enzyme activity was 6.5 to 6.9 and did not require Ca(2+) for the activity. Two molecular forms with enzyme activity were identified by size-exclusion chromatography and the molecular masses of the two enzymes were estimated to be 98 and 52 kDa. Two enzyme activities were strongly inhibited by Hg(2+), Cu(2+) and trypsin inhibitors such as TLCK, antipain and leupeptin. It cleaved the substrate, Boc-Arg-Val-Arg-Arg-MCA, between the dibasic sequence Arg-Arg, and needed a support of aminopeptidase B-like enzyme activity for the liberation of 7-amino-4-methylcoumarin. These results suggest that the enzyme is transported in rat sciatic nerves and involved in the post-translational processing of precursor proteins under the anterograde axonal transport. But there is absolutely no evidence for a role in precursor processing and such a putative role is purely speculative.     

离子束注入提高Trichosporon lactis T立体拆分布洛芬水平的研究

以从土壤中分离筛选到的可立体选择性水解布洛芬乙酯生成S-布洛芬的菌株Trichosporon lactisT为出发菌株,对其进行能量30KeV,剂量1×1015~5×1015ions/cm2的低能N 注入,筛选立体选择性水解布洛芬乙酯活性高的诱变株。菌株T.lactisT,在4×1015ions/cm2的诱变剂量下突变率最高,正向和负向突变率分别达32.9%和37.1%,因此选定该剂量为T.lactisT的最佳N 离子注入剂量。经离子束诱变,通过初筛和复筛,共筛选到7株水解布洛芬乙酯的高产菌株,其中诱变株K1培养24h时酶活力比出发菌株T高50%,且具有较好的遗传稳定性。将菌株K1和出发菌株T培养24h,分别加入布洛芬乙酯水解24h,二者水解布洛芬乙酯生成S-布洛芬旋光度均为 54.1°,对映体过量值ee%均为98%,K1菌株水解的产量达6.96g/L,而出发株仅为4.24g/L。     

Purification and characterization of a cell wall peptidase from Lactococcus lactis subsp. cremoris IMN-C12.

A peptidase from the cell wall fraction of Lactococcus lactis subsp. cremoris IMN-C12 has been purified to homogeneity by hydrophobic interaction chromatography, two steps of anion-exchange chromatography, and gel filtration. The molecular mass of the purified enzyme was estimated to be 72 kDa by gel filtration and 23 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pI of 4.0, and it has the following N-terminal sequence from the 2nd to the 17th amino acid residues: -Arg-Leu-Arg-Arg-Leu-?-Val-Pro-Gly-Glu-Ileu-Val-Glu-Glu-Leu-Leu. The peptidase is most active at pH 5.8 and at 33 degrees C with trileucine as the substrate. Reducing agents such as dithiothreitol, beta-mercaptoethanol, and cysteine strongly stimulated enzyme activity, while p-chloromercuribenzoate had an inhibitory effect. Also, metal chelators lowered the peptidase activity, which could not be restored with Ca2+ and Mg2+. The divalent cations Cu2+, Zn2+, Fe2+, and Hg2+ completely inhibited peptidase activity. The peptidase is capable of hydrolyzing tripeptides and some dipeptides, with a preference for peptides containing leucine and with the highest activity towards the tripeptides Leu-Leu-Leu, Leu-Trp-Leu, and Ala-Leu-Leu, which were hydrolyzed with Kms of 0.37, 0.18, and 0.61 mM, respectively.     

Selection of functional signal peptide cleavage sites from a library of random sequences.

The export of proteins to the periplasmic compartment of bacterial cells is mediated by an amino-terminal signal peptide. After transport, the signal peptide is cleaved by a processing enzyme, signal peptidase I. A comparison of the cleavage sites of many exported proteins has identified a conserved feature of small, uncharged amino acids at positions -1 and -3 relative to the cleavage site. To determine experimentally the sequences required for efficient signal peptide cleavage, we simultaneously randomized the amino acid residues from positions -4 to +2 of the TEM-1 beta-lactamase enzyme to form a library of random sequences. Mutants that provide wild-type levels of ampicillin resistance were then selected from the random-sequence library. The sequences of 15 mutants indicated a bias towards small amino acids. The N-terminal amino acid sequence of the mature enzyme was determined for nine of the mutants to assign the new -1 and -3 residues. Alanine was present in the -1 position for all nine of these mutants, strongly supporting the importance of alanine at the -1 position. The amino acids at the -3 position were much less conserved but were consistent with the -3 rules derived from sequence comparisons. Compared with the wild type, two of the nine mutants have an altered cleavage position, suggesting that sequence is more important than position for processing of the signal peptide.     

Synthesis and structural insights into the binding mode of the albomycin δ1 core and its analogues in complex with their target aminoacyl-tRNA synthetase

Despite of proven efficacy and well tolerability, albomycin is not used clinically due to scarcity of material. Several attempts have been made to increase the production of albomycin by chemical or biochemical methods. In the current study, we have synthesized the active moiety of albomycin δ1 and investigated its binding mode to its molecular target seryl-trna synthetase (SerRS). In addition, isoleucyl and aspartyl congeners were prepared to investigate whether the albomycin scaffold can be extrapolated to target other aminoacyl-tRNA synthetases (aaRSs) from both class I and class II aaRSs, respectively. The synthesized analogues were evaluated for their ability to inhibit the corresponding aaRSs by an in vitro aminoacylation experiment using purified enzymes. It was observed that the diastereomer having the 5′S, 6′R-configuration (nucleoside numbering) as observed in the crystal structure, exhibits excellent inhibitory activity in contrast to poor activity of its companion 5′R,6′S-diasteromer obtained as byproduct during synthesis. Moreover, the albomycin core scaffold seems well tolerated for class II aaRSs inhibition compared with class I aaRSs. To understand this bias, we studied X-ray crystal structures of SerRS in complex with the albomycin δ1 core structure 14a, and AspRS in complex with compound 16a. Structural analysis clearly showed that diastereomer selectivity is attributed to the steric restraints of the active site of SerRS and AspRS.     

Identification of the sid outer membrane receptor protein in Salmonella typhimurium SL1027.

Summary A protein of molecular weight 78,000 daltons, missing in albomycin and phage ES18 resistant mutants, has been identified in the outer membrane of Salmonella typhimurium SL1027. Mutants with a tonB like resistance and overproduction of outer membrane proteins due to iron shortage were also isolated. The mutation which leads to the protein deficiency maps in the sid gene region, the mutation related to overproduction of proteins maps near trp. Although the S. typhimurium and the E. coli protein mediate translocation of the iron complex ferrichrome and the structurally analogous antibiotic albomycin through the outer membrane no cross-reactivity exists in binding the phages T5, T1 and ES18 or colicin M.     

Purification and characterization of a Cys-Gly hydrolase from the gastropod mollusk,Patella caerulea

A magnesium-dependent cysteinyl-glycine hydrolyzing enzyme from the gastropod mollusk Patella caerulea was purified to electrophoretic homogeneity through a simple and rapid purification protocol. The molecular masses of the native protein and the subunit suggest that the enzyme has a homohexameric structure. Structural data in combination with kinetic parameters determined with Cys-Gly and compared with Leu-Gly as a substrate, indicate that the purified enzyme is a member of the peptidase family M17. The finding that an enzyme of the peptidase family M17 is responsible also in mollusks for the breakdown of Cys-Gly confirms the important role of this peptidase family in the glutathione metabolism.     

Enterochelin System of Iron Transport in Escherichia coli: Mutations Affecting Ferric-Enterochelin Esterase

Three mutant strains of Escherichia coli have been isolated which are lacking ferric-enterochelin esterase activity. This enzyme catalyzes the hydrolysis of the enterochelin moiety of ferric-enterochelin to yield ultimately three molecules of N-2,3-dihydroxybenzoylserine. The mutants (designated fes(-)) were shown to be unaffected in enterochelin biosynthesis, capable of enterochelin-mediated iron uptake, and able to utilize ferric-dihydroxybenzoylserine complexes normally. When grown under iron-deficient conditions, however, they showed an absolute requirement for added iron or citrate, a phenotype characteristic of mutants defective in some part of the enterochelin system of iron uptake. These results support the theory that iron, taken up by the cell as ferric-enterochelin is only available for general cell metabolism after hydrolysis of the ligand by enterochelin esterase. The three fes(-) strains were shown to be affected in the B component of enterochelin esterase. The fesB gene which is probably the structural gene coding for component B of the esterase, was shown to be located at about minute 14 on the E. coli chromosome together with seven other genes involved in the enterochelin system of iron transport.     

Identification of genes involved in siderophore transport in Streptomyces coelicolor A3(2)

The potential iron siderophore transporter genes have been determined from the genome sequence of Streptomyces coelicolor A3(2). One of these gene clusters, cdtABC, was disrupted and characterized to determine its role in the uptake of the siderophores produced by S. coelicolor. Resistance to the siderophore-like antibiotics, salmycin and albomycin, was tested in the parent and cdtABC mutant, showing that the parent, but not the mutant, was sensitive to salmycin, while both were resistant to albomycin. Ferrioxamine competition assays against salmycin suggest that the uptake of salmycin is via a ferrioxamine transport system. However, Fe-55 ferrioxamine B uptake experiments did not reveal any difference between the parent and mutant. This suggests that CdtABC specifically transports salmycin, while ferrioxamine uptake maybe substituted by another transport system.