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.     

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.     

Characterization of a plasmid carrying the Escherichia coli K-12 entD, fepA, fes, and entF genes

Abstract The plasmid pMS101 carries Escherichia coli K-12 genes ( entD, fes, entF ) essential for enterochelin-mediated iron transport [Laird, A.J. and Young, I.G., Gene 11 (1980) 359–366]. We have further characterized pMS101 and shown that it also contains the gene ( fepA ) for the 81 000 Da outer membrane ferrienterochelin receptor. Subcloning experiments in conjunction with complementation and maxicell studies demonstrated the gene order to be entD fepA fes entF . The entF - and fes -encoded polypeptides were found to be 115 000 and 42 000-Da soluble proteins, respectively. Plasmid-borne enterochelin cluster genes were overexpressed in iron-deficient conditions and their products were undetectable under iron-replete conditions.     

Defective growth functions in mutants of Escherichia coli K12 lacking a major outer membrane protein.

Various properties of mutants of Escherichia coli K12 lacking specific outer membrane proteins have been studied. ompA mutants are shown to grow less well than their parent strains under a variety of growth conditions, and after completion of growth to enter a decline phase in which viability is lost and the cells become heavily piliated and clump. They are defective in the uptake of amino acids, whereas the uptakes of the larger transport substrates ferrienterochelin and cyanocobalamin (vitamin B12) are normal. These ompA mutants also grow poorly at 42 °C. The implications of these results are discussed in terms of the function of the ompA. gene product. No growth or uptake defects were observed for ompB or tsx mutants.     

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.     

Salmonella typhimurium IroN and FepA Proteins Mediate Uptake of Enterobactin but Differ in Their Specificity for Other Siderophores

Salmonella typhimurium possesses two outer membrane receptor proteins, IroN and FepA, which have been implicated in the uptake of enterobactin. To determine whether both receptors have identical substrate specificities, fepA and iroN mutants and a double mutant were characterized. While both receptors transported enterobactin, the uptake of corynebactin and myxochelin C was selectively mediated by IroN and FepA, respectively.     

Export of FepA::PhoA fusion proteins to the outer membrane of Escherichia coli K-12.

A library of fepA::phoA gene fusions was generated in order to study the structure and secretion of the Escherichia coli K-12 ferric enterobactin receptor, FepA. All of the fusion proteins contained various lengths of the amino-terminal portion of FepA fused in frame to the catalytic portion of bacterial alkaline phosphatase. Localization of FepA::PhoA fusion proteins in the cell envelope was dependent on the number of residues of mature FepA present at the amino terminus. Hybrids containing up to one-third of the amino-terminal portion of FepA fractionated with their periplasm, while those containing longer sequences of mature FepA were exported to the outer membrane. Outer membrane-localized fusion proteins expressed FepA sequences on the external face of the outer membrane and alkaline phosphatase moieties in the periplasmic space. From sequence determinations of the fepA::phoA fusion joints, residues within FepA which may be exposed on the periplasmic side of the outer membrane were identified.     

Targeted mutagenesis of five conserved tryptophan residues of LolB involved in membrane localization of Escherichia coli lipoproteins

LolB, catalyzing the last step of lipoprotein transfer from the inner to the outer membrane of Escherichia coli, is itself a lipoprotein anchored to the outer membrane. Five Trp residues of LolB are conserved among LolB homologs in Gram-negative bacteria. These Trp residues were mutagenized to obtain defective LolB mutants. Mutation of Trp at position 52 to Pro impaired the receptor activity and caused accumulation of the LolA-lipoprotein complex in the periplasm. Similar mutants were obtained for Trp at position 117. A mutant with Gly instead of Trp at position 148 retained the receptor activity but inhibited growth upon its overproduction. The outer membrane sorting of this mutant seemed to be defective, lipoprotein transfer thereby being perturbed when it was overproduced. Despite the strong conservation, no defective mutant for Trp at position 183 was obtained, and only weak mutants were isolated for Trp at position 18. Based on the crystal structure of LolB, the phenotypes of these mutants are discussed.     

Suppression of iron uptake deficiency in Escherichia coli K-12 by loss of two major outer membrane proteins.

A novel iron uptake system was observed in pseudorevertants of $\text{Escherichia}$,$\text{coli}$ strains defective in ferrienterochelin transport. The new system is unique in that it is an active transport system that does not utilize any known siderophore. Acquisition of the new uptake system occurs concomitantly with the loss of two major outer membrane proteins (b and c) believed to function as structural components of transmembrane pores.     

Uptake of ferrienterochelin by Escherichia coli: energy dependent stage of uptake.

The uptake of the siderophore-iron complex ferrienterochelin was found to be strongly dependent upon an energized membrane state, as demonstrated by its sensitivity to dinitrophenol, azide, and cyanide. Ferrienterochelin uptake may also be dependent upon phosphate bond energy, as indicated by sensitivity to arsenate and iodoacetic acid. Although the adenosine triphosphatase does not appear to be involved in this energy coupling mechanism, ferrienterochelin uptake was shown to be less dependent upon phosphate bond energy than was glutamine uptake. Sensitivity of ferrienterochelin uptake to osmotic shock was shown to be due to the release of a ferrienterochelin binding compound located in the outer membrane of the cells and probably identical to the colicin B receptor protein.     

Biochemical analysis of spontaneous fepA mutants of Escherichia coli

The fepA gene of Escherichia coli encodes the outer-membrane receptor protein for ferrienterobactin. Previous genetic studies indicated that fepA mutations occur frequently and suggested that most of the mutations were deletions. In this work seven spontaneous fepA mutations were analyzed by enzyme assay (enterobactin synthase and enterobactin esterase) and by DNA hybridization studies. In two strains, UT500 and UT700, the mutations were confined to the fepA gene. In the remaining mutants, the mutations were large deletions; in several cases, 27 kb or more of DNA had been lost. The deletions, all of which eliminated approximately the left half of the enterobactin gene cluster, extended from the vicinity of the fepC gene counterclockwise into the chromosome. A minimum of three clockwise endpoints were identified and at least two counterclockwise endpoints were detected. The variation in endpoints among the deletions argues against the involvement of a normal transposon in their formation. Also, unexpected homology was found between enterobactin gene cluster DNA and lacPOZ and pSC101.     

Biosynthesis and structure of lipopolysaccharide in an outer membrane-defective mutant of Escherichia coli J5.

Membrane-defective mutants of Escherichia coli J5 were isolated on the basis of supersensitivity to the antibiotic novobiocin. These mutants display an increased sensitivity to a wide range of antibiotics and to several dyes and detergents. In addition, several mutants leak the periplasmic enzymes, alkyline phosphatase and ribonuclease. This evidence indicates an outer membrane defect in these mutants. The inner and outer membranes of one mutant were separated and subjected to compositional analysis. A deficiency in galactose containing lipopolysaccharide in the outer membrane of the mutant was observed. Two possible causes of this deficiency were examined and discounted: defective galactose uptake into the cell, and defective translocation of lipopolysaccharide from the inner membrane. Extraction and chemical analysis of mutant and wild type lipopolysaccharides suggests that the mutant is defective in the enzyme which transfers glucose to the growing lipopolysaccharide core, UDPglucose transferase. Thus, the mutant's deficiency in galactose-containing lipopolysaccharide can be ascribed to the fact that addition of glucose to the lipopolysaccharide core is a prerequisite for galactose addition. The physiological implications of this alteration are discussed.     

Biosynthesis and structure of lipopolysaccharide in an outer membrane-defective mutant of Escherichia coli J5

Membrane-defective mutants of Escherichia coli J5 were isolated on the basis of supersensitivity to the antibiotic novobiocin. These mutants display an increased sensitivity to a wide range of antibiotics and to several dyes and detergents. In addition, several mutants leak the periplasmic enzymes, alkaline phosphatase and ribonuclease. This evidence indicates an outer membrane defect in these mutants. The inner and outer membranes of one mutant were separated and subjected to compositional analysis. A deficiency in galactose-containing lipopolysaccharide in the outer membrane of the mutant was observed. Two possible causes of this deficiency were examined and discounted: defective galactose uptake into the cell, and defective translocation of lipopolysaccharide from the inner membrane. Extraction and chemical analysis of mutant and wild type lipopolysaccharides suggests that the mutant is defective in the enzyme which transfers glucose to the growing lipopolysaccharide core, UDPglucose transferase. Thus, the mutant's deficiency in galactose-containing lipopolysaccharide can be ascribed to the fact that addition of glucose to the lipopolysaccharide core is a prerequisite for galactose addition. The physiological implications of this alteration are discussed.     

Escherichia coli K-12 F? mutants that form mating aggregates but form transconjugants with low frequencies

Summary We have isolated Escherichia coli F^– mutants which, when mated with either Hfr or F, can form stable mating aggregates well but produce transconjugants with reduced frequencies. Selection procedure and other tests rule out the possibility that they are Rec^– strains. These mutants can be classified into two types: type I mutants can induce conjugal DNA replication in the donor, yet form transconjugants poorly; whereas, type II mutants induce conjugal DNA replication with poor efficiencies in the donor. Further tests indicate that type I mutants are very sensitive to lethal zygosis and their membranes, both inner and outer, show alterations in protein composition, whereas type II mutants are insensitive to lethal zygosis, and have an obvious alteration in the protein composition of their outer membrane. These results suggest that type I is defective in transconjugant formation primarily due to a change in the inner membrane, whereas type II is defective in generating a mating signal, which induces donor conjugal DNA replication, due to an alteration in the outer membrane.     

Iron regulated genes of Salmonella enterica serovar Typhimurium in response to norepinephrine and the requirement of fepDGC for norepinephrine-enhanced growth

Catecholamines may stimulate enteric bacteria including the foodborne pathogen Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) by two mechanisms in vivo: as a quorum sensing signal and a supplier of iron. To identify genes of Salmonella Typhimurium that respond to norepinephrine, transposon mutagenesis and DNA microarray analysis were performed. Insertional mutations in the following genes decreased norepinephrine-enhanced growth: degS, entE, entF, fes, gpmA, hfq, STM3846. DNA microarray and real-time RT-PCR analyses revealed a decrease in the expression of several genes involved in iron acquisition and utilization during norepinephrine exposure, signifying the iron-limiting conditions of serum-SAPI minimal medium and the siderophore-like activity of norepinephrine. Unlike the wild-type parent strain, growth of neither a fepA iroN cirA mutant nor a fepC mutant, harboring deletional mutations in the outer and inner membrane transporters of enterochelin, respectively, was enhanced by norepinephrine. However, growth of the fepC and the fepA iroN cirA mutants could be rescued by an alternative siderophore, ferrioxamine E, further validating the role of norepinephrine in supplying the organism with iron via the catecholate-specific iron transport system. Contrary to previous reports using small animal models, the fepA iroN cirA mutant of Salmonella Typhimurium colonized the swine gastrointestinal tract, as did the fepC mutant.     

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.     

Role of protein F in maintaining structural integrity of the Pseudomonas aeruginosa outer membrane.

To investigate the functional role of protein F of the outer membrane of Pseudomonas aeruginosa, we isolated mutants devoid of protein F, and the defective gene was transferred to a wild-type strain by plasmid FP5-mediated conjugation. Chemical analyses of the protein F-deficient outer membrane revealed that the amount of outer membrane protein was reduced to 72 to 74% of that of the protein F-sufficient strain and that lipopolysaccharides and phospholipids increased to 117 to 123% and 135 to 136%, respectively. The mutants and the transconjugant showed the following characteristics: (i) growth rates of protein F-deficient strains in low-osmolarity medium (e.g., L broth containing 0.1% NaCl) were less than 1/10 the rate of the protein F-sufficient strain; (ii) protein F-deficient cells were rounded, and the outer membrane formed large protruded blebs; and (iii) the outer membrane became physically fragile, since a significant amount of periplasmic proteins leaked out and the cells became highly sensitive to osmotic shock. The results suggested that protein F plays an important role in morphogenesis and in maintaining the integrity of the outer membrane. Determination of the diffusion rates of saccharides and beta-lactam antibiotics showed that the protein F-deficient outer membrane had no detectable transport defect compared with the protein F-sufficient outer membrane. The MICs of antibiotics for the protein F-deficient strains were nearly identical to those for the protein F-sufficient strain.