Outer Membrane-Dependent Transport Systems in Escherichia coli: Effect of Repression or Cessation of Colicin Receptor Synthesis on Colicin Receptor Activities

Proteins in the outer membrane of gram-negative bacteria serve as general porins or as receptors for specific nutrient transport systems. Many of these proteins are also used as receptors initiating the processes of colicin or phage binding and uptake. The functional activities of several outer membrane proteins in Escherichia coli K-12 were followed after cessation or repression of their synthesis. Cessation of receptor synthesis was accomplished with a thermolabile suppressor activity acting on amber mutations in btuB (encoding the receptor for vitamin B(12), the E colicins, and phage BF23) and in fepA (encoding the receptor for ferric enterochelin and colicins B and D). After cessation of receptor synthesis, cells rapidly became insensitive to the colicins using that receptor. Treatment with spectinomycin or rifampin blocked appearance of insensitive cells and even increased susceptibility to colicin E1. Insensitivity to phage BF23 appeared only after a lag of about one division time, and the receptors remained functional for B(12) uptake throughout. Therefore, possession of receptor is insufficient for colicin sensitivity, and some interaction of receptor with subsequent uptake components is indicated. Another example of physiological alteration of colicin sensitivity is the protection against many of the tonB-dependent colicins afforded by provision of iron-supplying siderophores. The rate of acquisition of this nonspecific protection was found to be consistent with the repression of receptor synthesis, rather than through direct and immediate effects on the tonB product or other components of colicin uptake or action.     

Functional stability of the bfe and tonB gene products in Escherichia coli.

The expression of several functional properties of the products of the bfe and tonB genes in Escherichia coli was measured after the specific termination of the synthesis of the products of these genes. This was accomplished by the use of a temperature-sensitive amber suppressor mutation, which allowed control, by manipulation of the growth temperature, of the level of product formed from suppressible mutant alleles of the bfe or tonB gene. The bfe product is an outer membrane receptor protein for vitamin B12, the E-colicins, and bacteriophage BF23. The identity of the tonB product is unknown, but it is necessary for a subsequent step of uptake of vitamin B12, iron chelates, all of the group B colicins, and bacteriophages T1 and phi 80. Results from a different experimental system had shown that the termination of expression of the bfe locus was rapidly followed by loss of sensitivity to colicins E2 and E3 and, subsequently, to bacteriophage BF23. This was confirmed with this experimental system. Receptors that were no longer functional for colicin or phage uptake remained fully effective for B12 uptake, showing that receptors are stable on the cell surface. This supports previous contentions for the presence of different functional states for colicin receptors. The functional properties of the tonB product, measured by B12 uptake or sensitivity to the group B colicin D, were unstable, declining extensively after cessation of its synthesis.     

Suppression of the btuB451 mutation by mutations in the tonB gene suggests a direct interaction between TonB and TonB-dependent receptor proteins in the outer membrane of Escherichia coli

In cells of Escherichia coli, the function of the tonB gene is needed for energy-dependent transport processes mediated by the outer-membrane receptors for iron siderophore complexes and vitamin B12. The btuB451 mutation has the same effect on vitamin B12 transport as does a tonB mutation. When a btuB451 strain carried a plasmid with the intact tonB gene, partial revertant strains were isolated which had acquired the ability to grow on 5 nM vitamin B12. This suppression activity was associated with the plasmid, suggesting that a mutation within the tonB gene on the plasmid allowed the mutant BtuB receptor to function in the transport of the vitamin. The nucleotide sequence of the entire tonB gene of ten independently isolated suppressing plasmids was determined. Only a single nucleotide change had occurred in each of the cases. The same codon was always affected resulting in the conversion of glutamine-165 to a leucine in seven of the ten isolates and to a lysine in the other three. The phenotype of strains carrying both types of altered tonB genes showed the retention of their function for other TonB-dependent processes in addition to their suppressor properties with respect to the btuB451 mutation. The fact that mutations suppressing the btuB451 mutation occurred in the tonB gene suggests that there is a direct interaction between TonB and TonB-dependent receptors in the outer membrane of E. coli.     

Relation of cell growth and colicin tolerance to vitamin B12 uptake in Escherichia coli.

The uptake of vitamin B12 was measured in cells of Escherichia coli whose growth had been inhibited by any of a variety of treatments. In all cases, the secondary, energy-dependent phase of B12 uptake was depressed in proportion to the decrease in growth rate, but uptake was constant in cells growing logarithmically at different rates. The depression of B12 uptake activity was independent of the site of cell metabolism affected by the inhibitor or by its effect on cell viability, and was both more rapid and of greater degree than the effects on the uptake of any of the six amino acids tested. The decline was not affected by inhibitors of either cell division or proteolysis and was manifested without any apparent decrease in the surface B12 binding activity. Transport activity was rapidly regained upon reversal of the inhibition of protein synthesis. Prompted by this response, the uptake of B12 was contrasted to the apparent uptake of the E colicins, which share the same outer membrane receptor. Sensitivity to colicin E1, measured by its inhibition of proline uptake, was not affected by growth inhibition by antibiotic treatment. Finally, there was no specific depression of B12 uptake in cells rendered colicin tolerant either by mutation or as a consequence of phage f1 infection.     

Transport of vitamin B12 in tonB mutants of Escherichia coli.

It is known that the tonB mutation in Escherichia coli is responsible for a defect in the transport of iron chelates. These are transported by systems that involve outer membrane components. We found that tonB mutants were also deficient in the secondary, energy-dependent phase of vitamin B12 transport, although the mutants have normal levels of B12 receptors on their cell surface. In addition, tonB mutants derived from vitamin B12 auxotrophs required elevated levels of B12 for normal growth. Maltose uptake, mediated by another transport system involving an outer membrane component, was unaffected by the tonB mutation.     

Genetic analysis of components involved in vitamin B12 uptake in Escherichia coli.

The products of three genes are involved in cyanocobalamin (B(12)) uptake in Escherichia coli. btuB (formerly bfe), located at min 88 on the Escherichia coli linkage map, codes for a protein component of the outer membrane which serves as receptor for B(12), the E colicins, and bacteriophage BF23. Four phenotypic classes of mutants varying in response to these agents were found to carry mutations that, based on complementation and reversion analyses, reside in the single btuB cistron. In one mutant class, ligand binding to the receptor appeared to be normal, but subsequent B(12) uptake was defective. The level of receptor and rate of uptake were responsive to btuB gene dosage. Previous studies showed that the tonB product was necessary for energy-dependent B(12) uptake but not for its binding. Other than those in tonB, no mutations that conferred insensitivity to group B colicins affected B(12) utilization. The requirement for the btuB and tonB products could be bypassed by elevated levels of B(12) (>1 muM) or by mutations compromising the integrity of the outer membrane as a permeability barrier. Utilization of elevated B(12) concentrations in strains lacking the btuB-tonB uptake system was dependent on the function of the btuC product. This gene was located at 37.7 min on the linkage map, with the order pps-btuC-pheS. Strains altered in btuC but with an intact btuB-tonB system were only slightly impaired in B(12) utilization, being defective in its accumulation. This defect was manifested as inability to retain B(12), such that intracellular label was almost completely lost by exchange or efflux. It is proposed that btuC encodes a transport system for B(12) in the periplasm.     

Point mutations in a conserved region (TonB box) of Escherichia coli outer membrane protein BtuB affect vitamin B12 transport.

Uptake of cobalamins and iron chelates in Escherichia coli K-12 is dependent on specific outer membrane transport proteins and the energy-coupling function provided by the TonB protein. The btuB product is the outer membrane receptor for cobalamins, bacteriophage BF23, and the E colicins. A short sequence near the amino terminus of mature BtuB, previously called the TonB box, is conserved in all tonB-dependent receptors and colicins and is the site of the btuB451 mutation (Leu-8----Pro), which prevents energy-coupled cobalamin uptake. This phenotype is partially suppressed by certain mutations in tonB. To examine the role of individual amino acids in the TonB box of BtuB, more than 30 amino acid substitutions in residues 6 to 13 were generated by doped oligonucleotide-directed mutagenesis. Many of the mutations affecting each amino acid did not impair transport activity, although some substitutions reduced cobalamin uptake and the Leu-8----Pro and Val-10----Gly alleles were completely inactive. To test whether the btuB451 mutation affects only cobalamin transport, a hybrid gene was constructed which encodes the signal sequence and first 39 residues of BtuB fused to the bulk of the ferrienterobactin receptor FepA (residues 26 to 723). This hybrid protein conferred all FepA functions but no BtuB functions. The presence of the btuB451 mutation in this fusion gene eliminated all of its tonB-coupled reactions, showing that the TonB box of FepA could be replaced by that from BtuB. These results suggest that the TonB-box region of BtuB is involved in active transport in a manner dependent not on the identity of specific side chains but on the local secondary structure.     

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.     

The tonB gene of Serratia marcescens: sequence, activity and partial complementation of Escherichia coli tonB mutants

The TonB protein plays a key role in the energy-coupled transport of iron siderophores, of vitamin B12, and of colicins of the B-group across the outer membrane of Escherichia coli. In order to obtain more data about which of its particular amino acid sequences are necessary for TonB function, we have cloned and sequenced the tonB gene of Serratia marcescens. The nucleotide sequence predicts an amino acid sequence of 247 residues (Mr 27,389), which is unusually proline-rich and contains the tandem sequences (Glu-Pro)5 and (Lys-Pro)5. In contrast to the TonB proteins of E. coli and Salmonella typhimurium, translation of the S. marcescens TonB protein starts at the first methionine residue of the open reading frame, which is the only amino acid removed during TonB maturation and export. Only the N-terminal sequence is hydrophobic, suggesting its involvement in anchoring the TonB protein to the cytoplasmic membrane. The S. marcescens tonB gene complemented an E. coli tonB mutant with regard to uptake of iron siderophores, and sensitivity to phages T1 and phi 80, and to colicins B and M. However, an E. coli tonB mutant transformed with the S. marcescens tonB gene remained resistant to colicins Ia and Ib, to colicin B derivatives carrying the amino acid replacements Val/Ala and Val/Gly at position 20 in the TonB box, and they exhibited a tenfold lower activity with colicin D. In addition, the S. marcescens TonB protein did not restore T1 sensitivity of an E. coli exbB tolQ double mutant, as has been found for the overexpressed E. coli TonB protein, indicating a lower activity of the S. marcescens TonB protein. Although the S. marcescens TonB protein was less prone to proteolytic degradation, it was stabilized in E. coli by the ExbBD proteins. In E. coli, TonB activity of S. marcescens depended either on the ExbBD or the TolQR activities.     

Repression of synthesis of the vitamin B12 receptor in Escherichia coli.

Growth of Escherichia coli K-12 strains in the presence of the vitamin cyanocobalamin (B12) resulted in an 80 to 90% reduction in B12 uptake activity of washed cells. Coincident with the decline in uptake activity was the depression of B12-binding activity in energy-poisoned cells, suggesting that growth in B12 resulted in the repression of synthesis of the B12 receptor protein in the outer membrane. Growth in the presence of B12 led to marked reduction in sensitivity to the E colicins, whose adsorption to cells requires the B12 receptor, and to a decrease in the amount of a band on electropherograms of outer membrane proteins. That polypeptide was also missing from mutants altered at btuB, the locus encoding the B12 receptor. Addition of B12 to growing cultures resulted in the exponential decline in specific activity of B12 uptake, as expected for dilution of functional receptors by further growth. Repression of receptor synthesis appears to be regulated by the level of intracellular, rather than extracellular, B12 and is separate from the regulation of the methionine biosynthetic pathway. Mutants altered in btuC, which are defective in accumulation and retention of B12, exhibit a much lower degree of repressibility.     

ExbBD-dependent transport of maltodextrins through the novel MalA protein across the outer membrane of Caulobacter crescentus

Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe(3+) and vitamin B(12)-the substrates hitherto known to be transported by TonB-dependent transporters-the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [(14)C]maltodextrins from [(14)C]maltose to [(14)C]maltopentaose. [(14)C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a K(d) of 0.2 microM, while the second transport had a K(d) of 5 microM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 microM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [(14)C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe(3+)-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe(3+)-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamB mutant, which suggests a low porin activity in C. crescentus. The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with K(d) values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B(12) and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin B(12) has been demonstrated.     

Genetic suppression demonstrates interaction of TonB protein with outer membrane transport proteins in Escherichia coli.

Energy-coupled reactions of the Escherichia coli outer membrane transport proteins BtuB and Cir require the tonB product. Some point mutations in a region of btuB and cir that is highly conserved in TonB-dependent transport proteins led to loss of TonB-coupled uptake of vitamin B12 and colicin Ia, whereas binding was unaffected. Most other point mutations in this region had no detectable effect on transport activity. Mutations in tonB that suppressed the transport defect phenotype of these btuB mutations were isolated. All carried changes of glutamine 165 to leucine, lysine, or proline. The various tonB mutations differed markedly in their suppression activities on different btuB or cir mutations. This allele specificity of suppression indicates that TonB interacts directly with the outer membrane transport proteins in a manner that recognizes the local conformation but not specific side chains within this conserved region. An effect of the context of the remainder of the protein was seen, since the same substitution (valine 10----glycine) in btuB and cir responded differently to the suppressors. This finding supports the proposal that TonB interacts with more of the transport proteins than the first conserved domain alone.     

Siderophore protection against colicins M, B, V, and Ia in Escherichia coli.

A variety of natural and synthetic siderophores capable of supporting the growth of Escherichia coli K-12 on iron-limited media also protect strain RW193+ (tonA+ ent-) from the killing action of colicins B, V, and Ia. Protective activity falls into two categories. The first, characteristic of enterobactin protection against colicin B and ferrichrome protection against colicin M, has properties of a specific receptor competition between the siderophore and the colicin. Thus, enterobactin specifically protects against colicin B in fes- mutants (able to accumulate but unable to utilize enterobactin) as predicted by our proposal that the colicin B receptor functions in the specific binding for uptake of enterobactin (Wayne and Neilands, 1975). Similarly ferrichrome specifically protects against colicin M in SidA mutants (defective in hydroxamate siderophore utilization). The second category of protective response, characteristic of the more general siderophore inhibition of colicins B, V, and Ia, requires the availability or metabolism of siderophore iron. Thus, enterobactin protects against colicins V and Ia, but only when the colicin indicator strain is fes+, and hydroxamate siderophores inhibit colicins B, V, and Ia, but only when the colicin indicator strain is SidA+. Moreover, ferrichrome inhibits colicins B, V, and Ia, yet chromium (III) deferriferrichrome is inactive, and ferrichrome itself does not prevent adsorption of colicin Ia receptor material in vitro. Although the nonspecific protection against colicins B, V, and Ia requires iron, the availability of siderophore iron for cell growth is not sufficient to bring about protection. None of the siderophores tested protect cells against the killing action of colicin E1 or K, or against the energy poisons azide, 2, 4-dinitrophenol, and carbonylcyanide m-chlorophenylhydrazone. We suggest that nonspecific siderophore protection against colicins B, V, and Ia may be due either to an induction of membrane alterations in response to siderophore iron metabolism or to a direct interference by siderophore iron with some unknown step in colicin action subsequent to adsorption.     

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.     

Import of the transfer RNase colicin D requires site-specific interaction with the energy-transducing protein TonB

The transfer RNase colicin D and ionophoric colicin B appropriate the outer membrane iron siderophore receptor FepA and share a common translocation requirement for the TonB pathway to cross the outer membrane. Despite the almost identical sequences of the N-terminal domains required for the translocation of colicins D and B, two spontaneous tonB mutations (Arg158Ser and Pro161Leu) completely abolished colicin D toxicity but did not affect either the sensitivity to other colicins or the FepA-dependent siderophore uptake capacity. The sensitivity to colicin D of both tonB mutants was fully restored by specific suppressor mutations in the TonB box of colicin D, at Ser18(Thr) and Met19(Ile), respectively. This demonstrates that the interaction of colicin D with TonB is critically dependent on certain residues close to position 160 in TonB and on the side chains of certain residues in the TonB box of colicin D. The effect of introducing the TonB boxes from other TonB-dependent receptors and colicins into colicins D and B was studied. The results of these and other changes in the two TonB boxes show that the role of residues at positions 18 and 19 in colicin D is strongly modulated by other nearby and/or distant residues and that the overall function of colicin D is much more dependent on the interaction with TonB involving the TonB box than is the function of colicin B.     

Energy-coupled colicin transport through the outer membrane of Escherichia coli K-12: mutated TonB proteins alter receptor activities and colicin uptake

Abstract The current model of TonB-dependent colicin transport through the outer membrane of Escherichia coli proposes initial binding to receptor proteins, vectorial release from the receptors and uptake into the periplasm from where the colicins, according to their action, insert into the cytoplasmic membrane or enter the cytoplasm. The uptake is energy-dependent and the TonB protein interacts with the receptors as well as with the colicins. In this paper we have studied the uptake of colicins B and Ia, both pore-forming colicins, into various tonB point mutants. Colicin Ia resistance of the tonB mutant (G186D, R204H) was consistent with a defective Cir receptor-TonB interaction while colicin Ia resistance of E. coli expressing TonB of Serratia marcescens , or TonB of E. coli carrying a C-terminal fragment of the S. marcescens TonB, seemed to be caused by an impaired colicin Ia-TonB interaction. In contrast, E. coli tonB (G174R, V178I) was sensitive to colicin Ia and resistant to colicin B unless TonB, ExbB and ExbD were overproduced which resulted in colicin B sensitivity. The differential effects of tonB mutations indicate differences in the interaction of TonB with receptors and colicins.     

Transport of vitamin B12 in Escherichia coli: cloning of the btuCD region.

The transport of vitamin B12 in Escherichia coli requires a specific vitamin B12 receptor protein in the outer membrane and the tonB gene product. In addition, the btuC gene, located at min 38 on the genetic map, has been found to influence vitamin B12 uptake or utilization. The btuC function is required for the growth response to vitamin B12 when the outer membrane transport process (btuB or tonB function) is defective. However, even in a wild-type strain, btuC is required for proper transport of vitamin B12. Additional mutations in the vicinity of btuC were isolated as lac fusions that produced a phenotype similar to that of a btuC mutant. The btuC region was cloned by selection for complementation of a btuC mutation. Complementation testing with plasmids carrying various deletions or transposon Tn1000 insertions demonstrated that the new mutations defined a separate, independently expressed locus, termed btuD. The coding regions for both genes were identified on a 3.4-kilobase HindIII-HincII fragment and were 800 to 1,000 base pairs in length. They were separated by a 600- to 800-base-pair region. The gene order in this portion of the chromosome map was found to be pps-zdh-3::Tn10-btuD-btuC-pheS. Expression of beta-galactosidase in the btuD-lac fusion-bearing strains, whether proficient or defective in vitamin B12 transport, was not regulated by the presence of vitamin B12 in the growth medium.     

Gab1 mediates neurite outgrowth, DNA synthesis, and survival in PC12 cells

The Gab1-docking protein has been shown to regulate phosphatidylinositol 3-kinase PI3K activity and potentiate nerve growth factor (NGF)-induced survival in PC12 cells. Here, we investigated the potential of Gab1 to induce neurite outgrowth and DNA synthesis, two other important aspects of NGF-induced neuronal differentiation of PC12 cells and NGF-independent survival. We generated a recombinant adenovirus encoding hemagglutinin (HA)-epitope-tagged Gab1 and expressed this protein in PC12 cells. HA-Gab1 was constitutively tyrosine-phosphorylated in PC12 cells and induced the phosphorylation of Akt/protein kinase B and p44/42 mitogen-activated protein kinase. HA-Gab1-stimulated a 10-fold increase in neurite outgrowth in the absence of NGF and a 5-fold increase in NGF-induced neurite outgrowth. HA-Gab1 also stimulated DNA synthesis and caused NGF-independent survival in PC12 cells. Finally, we found that HA-Gab1-induced neuritogenesis was completely suppressed by pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) activity and 50% suppressed by inhibition of PI3K activity. In contrast, HA-Gab1-stimulated cell survival was efficiently suppressed only by inhibition of both PI3K and MEK activities. These results indicate that Gab1 is capable of mediating differentiation, DNA synthesis, and cell survival and uses both PI3K and MEK signaling pathways to achieve its effects.     

DNA homology between siderophore genes from fluorescent pseudomonads.

Many species of pseudomonads produce fluorescent siderophores involved in iron uptake. We have investigated the DNA homology between the siderophore synthesis genes of an opportunist animal pathogen, Pseudomonas aeruginosa, and three plant-associated species Pseudomonas syringae, Pseudomonas putida and Pseudomonas sp. B10. There is extensive homology between the DNA from the different species, consistent with the suggestion that the different siderophore synthesis genes have evolved from the same ancestral set of genes. The existence of DNA homology allowed us to clone some of the siderophore synthesis genes from P. aeruginosa, and genetic mapping indicates that the cloned DNA lies in a locus previously identified as being involved in siderophore production.