Regulatory architecture of the iron-regulated fepD-ybdA bidirectional promoter region in Escherichia coli.

The overlapping and opposing promoter elements for the Escherichia coli fepDGC operon and the ybdA gene (encoding a 43-kDa cytoplasmic membrane protein) within the enterobactin gene cluster were investigated by measuring the effects of site-specific mutations on transcript levels and on expression of reporter genes in a bidirectional transcriptional fusion vector. Primary promoter structures for the opposing transcripts overlapped extensively such that their -10 sequences were almost directly opposed on the two strands of the DNA helix and their +1 transcription start sites were only 23 bp apart. Relative to the E. coli consensus sequence, both promoters were poorly conserved at the -35 position and mutations which strengthened the -35 element of either promoter significantly enhanced its transcription, decreased that of the opposing promoter, and dramatically altered iron-mediated regulation of expression. Both the fepD and ybdA primary promoters were shown to require a 5'-TGn-3' upstream extension of their -10 elements for optimal activities. Secondary promoters were identified for both fepD and ybdA, and their contributions to the overall expression levels were evaluated in these dual expression vector constructs. The data provided strong evidence that the architecture of the regulatory elements within the overlapping fepD and ybdA promoters is configured such that there is a direct competition for binding RNA polymerase and that the expression levels at these promoters are influenced not only by the activity of the opposing promoters but also by additional promoter sequence elements and perhaps accessory regulatory factors. Iron-mediated regulation of these promoters through the repressor protein Fur is a consequence of the relative promoter strengths and the position of an operator site that consists of two overlapping Fur-binding sequences in this compact regulatory region.

Heterogeneous changes in action potential and intracellular Ca2+ in left ventricular myocyte sub-types from rabbits with heart failure.

OBJECTIVE: Myocardial cellular electrophysiology and intracellular Ca2+ regulation are altered in heart failure. The extent of these changes may vary within the layers of the ventricular wall. To examine this, cell size, action potential and intracellular Ca2+ transient characteristics (Fura-2) were measured in single cardiac myocytes from sub-epicardial, mid-myocardial, and sub-endocardial regions of the left ventricle of rabbits with heart failure. METHODS: Myocytes were isolated from animals with heart failure induced by chronic coronary artery ligation and from sham operated controls. Trans-membrane potential was measured using high resistance microelectrodes electrodes (30 M omega; 2 M KC1). Fura-2 was loaded into cells by incubation with the AM form. Subsequent fluorescence measurements were used to measure intracellular Ca2+ concentration at a range of stimulus frequencies. RESULTS: Resting cell length was significantly greater in the heart failure group; approximately 115% of control values in sub-epicardial and mid-myocardial cells, and approximately 108% in sub-endocardial cells. Using criteria described by previous studies on other mammalian hearts, functional M cells were identified by a higher maximum rate of depolarisation and longer action potential duration at 90% repolarisation (APD90) compared to the two other myocyte sub-types. In the heart failure group, APD90 and Ca2+ transient duration (CaD50) were prolonged in sub-epicardial and M cells but shortened in sub-endocardial myocytes. These changes were significant at lower stimulus frequencies, but the relative effect diminished at higher frequencies (3 Hz). Peak systolic [Ca2+] was reduced in sub-epicardial and M cells but increased in sub-endocardial cells in the heart failure group compared to controls. At higher stimulus frequencies, end diastolic Ca2+ levels were lower in sub-epicardial cells but higher in sub-endocardial myocytes of the heart failure group compared with controls. In general, changes were greater in heart failure animals with more severe in vivo ventricular dysfunction (ejection fraction < or = 44%). CONCLUSIONS: Heart failure was associated with an increased cell size throughout the left ventricle, but the form of the changes in electrophysiology and Ca2+ transient were dependent on the myocyte sub-type. In particular sub-endocardial cells displayed markedly different changes compared to the other myocyte sub-types.

An amplifiable DNA region from the Mycoplasma hyorhinis genome.

A novel amplifiable genomic region that displays variability in the number of tandem copies of a 1,368-bp DNA sequence (designated RS-2) was discovered among individual clonal derivatives within Mycoplasma hyorhinis broth-grown cell populations. Clonal isolates representing variant subpopulations from the original broth culture were of a single size variant, and although continued culture under a variety of growth conditions did not result in further amplification of RS-2, evidence for deletion events which reduced RS-2 copy number, presumably by homologous recombination, was obtained. RS-2 homologous sequences were identified in all M. hyorhinis strains tested, but only the tissue culture-derived strains GDL-1 and GDL-2 showed variability in genomic dosage. The RS-2 nucleotide sequence established that each tandem copy is flanked by direct repeats of a 20-bp sequence and suggested a possible mechanism for its original duplication as the initial phase of a genetic amplification process. The coding strand was defined by PCR amplification of a reverse transcriptase-generated cDNA, and its sequence revealed that RS-2 encodes a 456-residue internal, highly cysteine-rich domain of a larger M. hyorhinis protein whose coding sequence initiates and terminates in unique genomic sequences several hundred base pairs from RS-2 on either side of it. Changes in RS-2 copy number maintain the reading frame, and therefore the coding capacity, for this predicted size-variant protein.

Promoter and operator determinants for fur-mediated iron regulation in the bidirectional fepA-fes control region of the Escherichia coli enterobactin gene system.

The fepA-entD and fes-entF operons in the enterobactin synthesis and transport system are divergently transcribed from overlapping promoters, and both are inhibited by the Fur repressor protein under iron-replete conditions. A plasmid harboring divergent fepA'-phoA and fes-entF'-'lacZ fusions, both under the control of this bidirectional regulatory region, was constructed for the purpose of monitoring changes in expression of the two operons simultaneously. Deletion analysis, site-directed mutagenesis, and primer extension were employed to define both a single promoter governing the expression of fes-entF and two tandemly arranged promoters giving rise to the opposing fepA-entD transcript. A single Fur-binding site that coordinately regulates the expression of all transcripts emanating from this control region was identified by in vitro protection from DNase I nicking. The substitution of one base pair in the Fur recognition sequence relieved Fur repression but did not change the in vitro affinity of Fur for its binding site. Additional mutations in a limited region outside of the promoter determinants for either transcript inhibited expression of both fes and fepA. These observations suggest a mechanism of Fur-mediated regulation in this compact control region that may involve other regulatory components.

Overexpression and purification of ferric enterobactin esterase from Escherichia coli. Demonstration of enzymatic hydrolysis of enterobactin and its iron complex.

The Escherichia coli ferric enterobactin esterase gene (fes) was cloned into the vector pGEM3Z under the control of the T7 gene 10 promoter and overexpressed to approximately 15% of the total cellular protein. The ferric enterobactin esterase (Fes) enzyme was purified as a 43-kDa monomer by gel filtration chromatography. Purified Fes preparations were examined for esterase activity on enterobactin and its metal complexes and for iron reduction from ferric complexes of enterobactin and 1,3,5-tris(N,N',N"-2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM), a structural analog lacking ester linkages. Fes effectively catalyzed the hydrolysis of both enterobactin and its ferric complex, exhibiting a 4-fold greater activity on the free ligand. It also cleaved the aluminum (III) complex at a rate similar to the ferric complex, suggesting that ester hydrolysis of the ligand backbone is independent of any reductive process associated with the bound metal. Ferrous iron was released from the enterobactin complex at a rate similar to ligand cleavage indicating that hydrolysis and iron reduction are tightly associated. However, no detectable release of ferrous iron from the MECAM complex implies that, with these in vitro preparations, metal reduction depends upon, and is subsequent to, the esterase activity of Fes. These observations are discussed in relation to studies which show that such enterobactin analogs can supply growth-promoting iron concentrations to E. coli.

A Mycoplasma hyorhinis protein with sequence similarities to nucleotide-binding enzymes.

We have determined the nucleotide (nt) and deduced amino acid (aa) sequence of a unique 115-kDa Mycoplasma hyorhinis protein (P115) with an N-terminal region containing a highly conserved consensus sequence characteristics of nt-binding domains of several ATPase and GTPase enzymes. However, P115 lacked additional conserved features characteristic of some classes of nt-binding proteins. Based on the hydropathy profile of the deduced aa sequence, the absence of a leader peptide, its exclusive partitioning into the hydrophilic phase during Triton X-114 phase fractionation of M. hyorhinis, and immunofluorescence analysis indicating no surface-exposed domains, it was concluded that P115 is a cytoplasmic protein lacking intrinsic membrane interaction. M. hyorhinis P115 appears to be a species-specific protein, since it was not detected in any other mycoplasmal or bacterial species examined with specific antibody or genomic probes. Since genetic systems for direct mutational analysis are currently unavailable in this organism, sequence analysis provides critical information in establishing the possible function of this protein. Moreover, the nt sequence encoding P115 reported here supports a previously proposed model, based on synthesis of P115-related proteins in Escherichia coli, suggesting that multiple polypeptide products can be generated from mycoplasma genes by promiscuous translation initiation in this heterologous expression system.

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.

Identification and mapping of an immunogenic region of Mycoplasma hyopneumoniae p65 surface lipoprotein expressed in Escherichia coli from a cloned genomic fragment.

A previously characterized lipid-modified amphiphilic surface protein of Mycoplasma hyopneumoniae, p65, has been defined by its reaction with a surface-binding monoclonal antibody (MAb) and by its exclusive partitioning into the detergent phase during Triton X-114 phase fractionation (K. S. Wise and M. F. Kim, J. Bacteriol. 169:5546-5555, 1987). In the current study, polyclonal mouse antibody (PAb) to gel-purified p65 was used to identify recombinant phage plaques expressing p65-related epitopes. Several characteristic partial tryptic fragments of p65 were recognized by both PAb and p65 and MAb to p65, but the PAb population specifically eluted from recombinant phage plaques bound only epitopes restricted to the largest of these fragments. Graded carboxypeptidase-Y digestion of intact M. hyopneumoniae generated C terminally truncated peptides that were recognized by PAb to p65 and MAb to p65, indicating that the C terminus and much of the adjoining region of p65 were present and accessible on the external face of the membrane. However, antibody eluted from recombinant phage plaques bound only to the largest truncated polypeptide, suggesting that a recombinant product corresponding to the C-terminal region of p65 was expressed in Escherichia coli. A 19-kilodalton recombinant protein (p19), which was recognized by PAb to p65 but not by MAb to p65, was detected in recombinant phage lysates. Serum antibodies from swine taken after, but not before, experimentally induced M. hyopneumoniae pneumonia preferentially recognized the native, amphiphilic p65 lipoprotein and also bound specifically to the p19 recombinant product. This confirmed that the p65 lipoprotein is a major immunogen of M. hyopneumoniae recognized during disease and identified its C-terminal region as an immunogenic domain.

Multiple translational products from a Mycoplasma hyorhinis gene expressed in Escherichia coli.

We analyzed protein expression from a cloned Mycoplasma hyorhinis genomic fragment that produces in Escherichia coli a set of related polypeptides of 110, 100, 65, and 55 kilodaltons from a coding region of just over 3.0 kilobases. Expression of these multiple products resulted from a mechanism operating at the translational level but not from truncation at UGA termination codons, which are known to encode tryptophan in several mycoplasma species. The structural relatedness of the proteins was demonstrated by two-dimensional tryptic peptic mapping, but their generation by posttranslational processing was ruled out by pulse-chase labeling analysis. Examination of proteins expressed from plasmid constructs and tryptic peptide analysis of these polypeptides and the original set of proteins revealed that they share carboxy-terminal regions, an observation inconsistent with truncation at UGA codons. Expression of proteins from this cloned fragment was not dependent on vector sequences and was observed when the coding region was placed under control of a T7 promoter, suggesting that all products were translated from a single message. Expression of related products in mycoplasmas was examined by immunoblot analysis of M. hyorhinis proteins with antiserum against overexpressed recombinant proteins. A single 115-kilodalton mycoplasma protein was detected, which is larger than any of the related proteins expressed in E. coli. Our analysis indicated that translation initiation sites are used in E. coli that are not active in mycoplasmas, thereby defining differences between the translational regulatory signals of mycoplasmas and eubacteria.

Regulation of divergent transcription from the iron-responsive fepB-entC promoter-operator regions in Escherichia coli.

Transcriptional linkage of the enterobactin gene cluster entCEBA (P15) was confirmed by ent-lacZ gene fusion analysis. Control sequences directing iron-regulated expression of this polycistronic message were localized to the fepB-entC bidirectional promoter region. Transcriptional initiation sites defined by primer extension analysis were located 103 base-pairs apart for the divergent fepB and entC messages. Within this divergent regulatory region, strongly consensus -35 and -10 promoter determinants and potential Fur repressor-binding sequences were identified. A vector containing divergently oriented indicator gene fusions was constructed to monitor regulatory effects of mutations within this iron-responsive control region. The fepB-entC promoter-operator elements were confirmed by mutation, using the dual gene fusion system in multicopy and low copy number states. Mutations in the -35 and -10 regions of the fepB and entC promoters that decreased their similarity to consensus resulted in reduced promoter activity. Mutations in the Fur-controlled operators reduced induction ratios (iron-deficient levels/iron-rich levels) for the respective fusion gene activities by approximately sevenfold. Although operator mutants retained some degree of inducibility, complete relief of repression was observed for double operator mutants, suggesting that only minor regulatory influence is exerted by Fur occupation of the opposing operator site. DNase I footprinting experiments were performed to characterize the sequence-specific Fur interactions at the operator sequences. At the fepB operator, a 31 base-pair Fur-protected region was identified, corresponding to positions -19 to +12 with respect to the transcriptional start site. Similarly, Fur protected a 31 base-pair region in entC, corresponding to positions +1 to +31 in the message. A contiguous and sequentially occupied secondary Fur-binding site in entC was protected at higher Fur concentrations, extending the protected region to +49, and sequestering the putative Shine-Dalgarno sequence. Operator positional effects and co-operativity are discussed.

The Escherichia coli enterobactin biosynthesis gene, entD: nucleotide sequence and membrane localization of its protein product.

The nucleotide sequence of the Escherichia coli enterobactin biosynthesis gene entD has been determined. entD specifies a predicted 23579 Dalton protein containing several helical regions, a transmembrane segment and one positively charged domain. The EntD polypeptide was overexpressed and identified in electrophoretic gels as a membrane protein. Although results of conventional membrane fractionation techniques were inconclusive, protease accessibility studies provided evidence that EntD domains are exposed on the inner leaflet of the cytoplasmic membrane. The presence of repetitive extragenic palindromic (REP) sequences within the fepA-entD intercistronic region was confirmed. Lack of a canonical promoter and an iron control region 5' to entD, along with RNA hybridization data, suggest that an iron-regulated transcript contains both fepA and entD.

Immune response to the iron-deprivation-induced proteins of Salmonella typhi in typhoid fever.

Iron starvation conditions limited the growth of Salmonella typhi, as evidenced by an increase in the lag phase of a culture and a decrease in the number of bacteria reached in the stationary phase. The analysis of the outer membrane of bacteria grown under these conditions identified new protein components with apparent molecular weights of 83,000, 78,000, and 69,000. The extent of induction of these proteins was regulated by increased iron deprivation. Immunoblot analysis showed that the serum of patients with typhoid fever exhibited an immunoglobulin G response to these iron-deprivation-induced proteins. The results of bioassays and DNA-DNA hybridization experiments indicated that pathogenic strains of S. typhi produced enterochelin but not aerobactin. Immunodetection with an anti-FepA antiserum confirmed that one of the induced proteins is the S. typhi analog of the Escherichia coli fepA gene product. These studies suggest a role for iron uptake in the pathogenesis of typhoid fever and confirm the immunogenicity of some of the outer membrane proteins of this pathogen.

Nucleotide sequence and transcriptional organization of the Escherichia coli enterobactin biosynthesis cistrons entB and entA.

The nucleotide sequence of a 2,137-base-pair DNA fragment expressing enterobactin biosynthesis functions defined the molecular boundaries and translational products of the entB and entA genes and identified a closely linked downstream open reading frame encoding an uncharacterized protein of approximately 15,000 daltons (P15). The sequence revealed that an independent protein-coding sequence corresponding to an EntG polypeptide was not situated in the genetic region between the entB and entA cistrons, to which the EntG- phonotype had been genetically localized. As a result, the biochemical nature of the EntG function in the biosynthetic pathway requires reevaluation. The EntA polypeptide displayed significant similarities at the amino acid level to the pyridine nucleotide-binding domains of several members of a family of alcohol-polyol-sugar dehydrogenase enzymes, consistent with its function as the enzyme catalyzing the final step of dihydroxybenzoate biosynthesis. An additional role for EntA in the isochorismate synthetase activity of EntC was strongly implicated by genetic evidence. Evidence from the nucleotide sequence of this region and newly constructed ent-lacZ fusion plasmids argues strongly that these genes are linked in an iron-regulated entCEBA (P15) polycistronic operon.

Transcriptional mapping and nucleotide sequence of the Escherichia coli fepA-fes enterobactin region. Identification of a unique iron-regulated bidirectional promoter.

The iron-controlled fepA and fes-entF transcripts from the Escherichia coli enterobactin gene complex are expressed divergently from a limited genetic region, thereby suggesting the existence of a single, possibly overlapping promoter junction for these genes. The nucleotide sequence of a 1,997-base pair HpaI fragment specific for this genetic region allowed for the identification of an 1,122-base pair open reading frame as the previously uncharacterized fes gene. Its product, Fes (approximately Mr 42,573) plays an essential but as yet ambiguous role in the release of ferric iron from the ligand. An additional small open reading frame of 216 nucleotides (encoding a potential product of calculated Mr 8,271) was also identified between fes and entF. A portion of the remaining nucleotide sequence defined a 320-base pair control region for both the fepA and fes-entF messages. Primer extension analyses placed the major in vivo transcription initiation sites to within 18 nucleotides of one another, thereby revealing a novel, extensively overlapping bidirectional promoter as well as long dual leader transcripts. This promoter region contains multiple overlapping nucleotide stretches which show strong homology to the consensus Fur repressor-binding sequence, forms of which are found in all E. coli iron-regulated promoters characterized to date.

Cluster of genes controlling synthesis and activation of 2,3-dihydroxybenzoic acid in production of enterobactin in Escherichia coli.

The Escherichia coli gene cluster encoding enzymatic activities responsible for the synthesis and activation of 2,3-dihydroxybenzoic acid in the formation of the catechol siderophore enterobactin was localized to a 4.2-kilobase chromosomal DNA fragment. Analysis of various subclones and transposon insertion mutations confirmed the previously suggested gene order as entEBG(AC) and provided evidence to suggest that these genes are organized as three independent transcriptional units, composed of entE, entBG, and entAC, with the entBG mRNA transcribed in a clockwise direction. Plasmid-specific protein expression in E. coli minicells identified EntE and EntB as 58,000- and 32,500-dalton proteins, respectively, while no protein corresponding to EntG was detected. The EntA and EntC enzymatic activities could not be separated by genetic or molecular studies. A small DNA fragment encoding both activities expressed a single 26,000-dalton polypeptide, suggesting that this protein is a multifunctional enzyme catalyzing two nonsequential reactions in the biosynthetic pathway. A protein of approximately 15,000 daltons appears to be encoded by the chromosomal region adjacent to the entAC gene, but no known function in enterobactin biosynthesis or transport can yet be ascribed to this polypeptide.

Genetic organization of multiple fep genes encoding ferric enterobactin transport functions in Escherichia coli.

Three genes were shown to provide functions specific for ferric enterobactin transport in Escherichia coli: fepA encoded the outer membrane receptor, fepB produced a periplasmic protein, and the fepC product was presumably a component of a cytoplasmic membrane permease system for this siderophore. A 10.6-kilobase-pair E. coli chromosomal EcoRI restriction fragment containing the fepB and fepC genes was isolated from a genomic library constructed in the vector pBR328. Both cistrons were localized on this clone (pITS24) by subcloning and deletion and insertion mutagenesis to positions that were separated by approximately 2.5 kilobases. Within this region, insertion mutations defining an additional ferric enterobactin transport gene (fepD) were isolated, and polarity effects from insertions into fepB suggested that fepD is encoded downstream on the same transcript. A 31,500-dalton FepC protein and a family of FepB polypeptides ranging from 34,000 to 37,000 daltons were identified in E. coli minicells, but the product of fepD was not detectable by this system. Another insertion mutation between entF and fepC was also shown to disrupt iron transport via enterobactin and thus defined the fepE locus; fepE weakly expressed a 43,000-dalton protein in minicells. It is proposed that these newly identified genes, fepD and fepE, provide functions which act in conjunction with the fepC product to form the ferric enterobactin-specific cytoplasmic membrane permease. An additional 44,000-dalton protein was identified and shown to be expressed from a gene that is situated between fepB and entE and that is transcribed in the direction opposite that of fepB. Although the function of this protein is uncharacterized, its membrane location suggests that it too may function in iron transport.

Physical and genetic characterization of cloned enterobactin genomic sequences from Escherichia coli K-12.

We have cloned genes responsible for enterobactin synthesis (entD) and transport (fepA,fes) from Escherichia coli K-12. Relevant recombinant plasmids enabled EntD- and transport-defective mutants to grow on iron-limiting medium. Subcloning and deletion analysis demonstrated that the gene order is entD-fepA-fes. Protein synthesis studies in minicells suggest that FepA is first translated as an Mr 84 000 precursor, which is subsequently cleaved to the active Mr 81 000 receptor; the fes gene product is an Mr 44 000 protein; no polypeptide has been identified as the entD gene product.

Regulation of enterobactin iron transport in Escherichia coli: characterization of ent::Mu d(Apr lac) operon fusions.

The vector Mu d(Apr lac) was utilized to construct operon fusions in the Escherichia coli enterobactin (ent) biosynthetic and transport genes. Enzyme assays indicated a 5- to 15-fold increase in the expression of beta-galactosidase when the fusion strains were grown under iron-deficient conditions. The polarity effects seen by Mu d insertions into entA, entC, and entE were consistent with a single operon, entA(CGB)E. The direction of transcription from iron-regulated promoters was determined by directional transfer of selected genetic markers after the insertion of F'ts114 lac+. Regulatory mutants were isolated in the fusion strains by the selection for constitutive expression of beta-galactosidase and the iron-regulated outer membrane proteins.

Role of iron in microbe-host interactions.

The ability of microorganisms that would like to live in or on mammalian hosts to acquire iron is a critical determinant of the host-parasite interaction. Despite the abundance of iron, its availability to microbes is restricted by the iron-binding and transport systems of the host. The successful commensal or pathogen therefore must express effective systems to compete for its iron. The acquisition of iron is thus essential, although not sufficient, for virulence. This review examines host and microbial iron-acquisition and transport mechanisms in an attempt to stimulate the reader's interest in potential "soft spots" that may be exploited prophylactically and therapeutically.

Kinetics of biosynthesis of iron-regulated membrane proteins in Escherichia coli.

Using biological iron chelators to control specifically iron availability to Escherichia coli K-12 in conjunction with radioactive pulse-labels, we examined the biosynthesis of six iron-regulated membrane proteins. Iron deprivation induced the synthesis of five proteins, which had molecular weights of 83,000 (83K), 81K (Fep), 78K (TonA), 74K (Cir), and 25K. The kinetics of induction were the same in entA and entA(+) strains, but were affected by the initial iron availability in the media. Iron-poor cells induced rapidly (half-time, 10 min), whereas iron-rich cells began induction after a lag and showed a slower induction half-time (30 min). Within this general pattern of induction after iron deprivation, several different kinetic patterns were apparent. The 83K, 81K, and 74K proteins were coordinately controlled under all of the conditions examined. The 78K and 25K proteins were regulated differently. The synthesis of a previously unrecognized 90K inner membrane protein was inhibited by iron deprivation and stimulated by iron repletion. Both ferrichrome and ferric enterobactin completely repressed 81K and 74K synthesis when the siderophores were supplied at concentrations of 5 muM in vivo (half-time, 2.5 min). At concentrations less than 5 muM, however, both siderophores repressed synthesis only temporarily; the duration of repression was proportional to the amount of ferric siderophore added. The half-lives of the 81K and 74K mRNAs, as measured by rifampin treatment, were 1.2 and 1.6 min, respectively. The results of this study suggest that enteric bacteria are capable of instantaneously detecting and reacting to fluctuations in the extracellular iron concentration and that they store iron during periods of iron repletion for utilization during periods of iron stress. Neither iron storage nor iron regulation of envelope protein synthesis is dependent on the ability of the bacteria to form heme.