Operon Fusion Research Articles

A mutation in the dsbA gene coding for periplasmic disulfide oxidoreductase reduces transcription of the Escherichia coli ompF gene

An insertion mutation in the Escherichia coli dsbA gene, coding for periplasmic disulfide oxidoreductase, dramatically reduces the level of OmpF porin protein in the cell envelope. Studies with chromosomal ompF-lacZ operon and gene fusions indicate that this is due to reduced ompF transcription. Identical effects resulted from growth in medium containing the reducing agent dithiothreitol, but the combined effects of the reducing agent and dsbA were no greater than the effects of each individually. The dsbA mutation did not prevent normal regulation of ompF transcription by the local anaesthetic procaine or by osmolarity. OmpF does not contain a cysteine residue, and the sole cysteine residue in the cytoplasmic membrane regulator of ompF transcription, EnvZ, is predicted to be located on the cytoplasmic side of the membrane, and is therefore unlikely to be involved in the effects of the dsbA mutation. The effects of the dsbA mutation and of the reducing agent on ompF transcription may be due to the failure to from an essential disulfide bond in an as yet unidentified envelope protein that affects ompF transcription.

Iron specificity of the Fur-dependent regulation of the biosynthesis of the manganese-containing superoxide dismutase in Escherichia coli.

The Fur protein, which regulates iron uptake in Escherichia coli, also represses the biosynthesis of the manganese-containing superoxide dismutase (MnSOD). A strain of E. coli bearing a lacZ fusion to the aerobactin operon was used to compare the metal specificities of the regulation of MnSOD and of aerobactin by Fur. Iron, but not manganese, acted as a corepressor of the Fur-dependent inhibition of MnSOD biosynthesis. The iron-mediated inhibition of MnSOD biosynthesis was dependent upon an intact fur locus, indicating that the effect of iron is mediated by the fur gene product. The suppression of the accumulation of MnSOD by iron, but not by manganese, was not due to destabilization of the MnSOD polypeptide by iron. Thus this effect of iron was also seen in a sodA::lacZ operon fusion in which the production of beta-galactosidase was regulated by the sodA promoter. In contrast, both iron and manganese served as corepressors of aerobactin biosynthesis. It thus appears that the effectiveness of specific metal cations to act as corepressors with Fur varies with the gene being regulated by the Fur-metal complex.

Molecular analysis of the phoH gene, belonging to the phosphate regulon in Escherichia coli.

By making operon fusions with lambda placMu53, we identified, cloned, and analyzed the phoH gene belonging to the phosphate (pho) regulon. We mapped the phoH gene at 23.6 min in the Escherichia coli genomic library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). Its nucleotide sequence revealed an open reading frame of 354 amino acids which contains sequences for nucleotide-binding motifs. From comparison of the DNA sequences, phoH was found to be identical to psiH, which had been identified as a phosphate starvation-inducible gene (W.W. Metcalf, P.M. Steed, and B.L. Wanner, J. Bacteriol. 172:3191-3200, 1990). The PhoH protein was overproduced by the T7 promoter system, identified as a protein of about 39 kDa, and purified. The amino-terminal amino acid sequence of the PhoH protein agreed with the one deduced from the DNA sequence. We demonstrated that PhoH has an ATP-binding activity by a photoaffinity labeling experiment. Two transcriptional initiation sites (P1 and P2) were identified by S1 nuclease mapping. The upstream P1 promoter contains a pho box, the conserved sequence shared by the pho regulon genes. The region containing the pho box was bound by PhoB protein, the transcriptional activator of the pho regulon, as revealed by footprinting. Regulation of phoH expression in vivo was studied by constructing plasmids containing transcriptional fusions of the phoH promoters with a promoterless gene for chloramphenicol acetyltransferase. Transcription from the P1 promoter required the phoB function and was induced by phosphate limitation, while transcription from the P2 promoter was independent of phoB and constitutive under tested conditions.

Interaction of six global transcription regulators in expression of manganese superoxide dismutase in Escherichia coli K-12.

Transcription of the sodA gene of Escherichia coli, which encodes manganese superoxide dismutase, is governed by six global regulators: the product of the soxRS locus (superoxide response) and mutated alleles of the soxQ locus (such as cfxB) act as activators; the products of the fur (ferric uptake regulation), arcA (aerobic regulation control), and fnr (fumarate nitrate reductase) genes and the integration host factor (IHF) negatively regulate sodA. The action of these effectors on the sodA promoter was investigated by using chromosomal sodA-lacZ operon fusions with intact or deleted promoters, different environmental conditions, and strains carrying different combinations of null mutations in the effector genes. The data allow us to assign target regions in the sodA promoter for activation by SoxRS and CfxB and for repression by Fur and ArcA. In aerobiosis, activation of sodA transcription by SoxRS was compatible with CfxB activation or Fur repression, whereas cfxB and fur controls were mutually exclusive. Repression by Fnr appeared, at least in part, to be ArcA dependent. IHF enhanced aerobic Fur repression, and in the absence of Fur, it enhanced anaerobic repression by ArcA. The DNA targets for Fur (encompassing the -35 region) and ArcA (from and downstream of the -35 region) appear to overlap, suggesting that Fur and ArcA repressions are mutually exclusive. Fur (in response to the iron pool) or ArcA, acting with Fnr and IHF (in response to the redox state of the cells), can block anaerobic sodA-lacZ expression with about equivalent efficiencies. The possible biological significance of this result is discussed.

Expression of theEscherichia coli ftsZgene: trials and tribulations of gene fusion studies

The ftsZ gene of Escherichia coli, which codes for an essential cell division protein, is subjected to multiple regulation, as shown in part with studies using an ftsZ::lacZ operon fusion located on phage lambda JFL100. Using this same fusion, we sought to isolate regulatory mutants overexpressing ftsZ by selecting mutants able to grow on lactose. One Lac+ mutant was obtained which overexpressed the ftsZ::lacZ fusion 70-fold. The mutation responsible for the overexpression lies in a new gene, cot, located near 56 min on the E. coli genetic map. The cot mutation probably affects the transcription of a chromosomal open reading frame, ORF1, lying downstream of the bioA gene and adjacent to the ftzZ::lacZ fusion of the lambda JFL100 prophage integrated at att lambda. Using an ftsZ84(Ts) strain, in which there was a double selection for overexpression of both ftsZ::lacZ and ftsZ+, no Lac+Tr mutants were obtained from 3.6 x 10(10) bacteria; the introduction of a mutL allele, increasing spontaneous base substitution mutation rates 75-fold, did not permit us to isolate such a mutant. We conclude that Lac+ ftsZ-constitutive mutations cannot be obtained in lambda JFL100 lysogens by a single base substitution.

Identification of the regulatory sequence of anaerobically expressed locus aeg-46.5

A newly identified anaerobically expressed locus, aeg-46.5, which is located at min 46.5 on Escherichia coli linkage map, was cloned and analyzed. The phenotype of this gene was studied by using a lacZ operon fusion. aeg-46.5 is induced anaerobically in the presence of nitrate in wild-type and narL cells. It is repressed by the narL gene product, as it showed derepressed anaerobic expression in narL mutant cells. We postulate that aeg-46.5 is subject to multiple regulatory systems, activation as a result of anaerobiosis, narL-independent nitrate-dependent activation, and narL-mediated repression. The regulatory region of aeg-46.5 was identified. A 304-bp DNA sequence which includes the regulatory elements was obtained, and the 5' end of aeg-46.5 mRNA was identified. It was verified that the anaerobic regulation of aeg-46.5 expression is controlled on the transcriptional level. Computer analysis predicted possible control sites for the NarL and FNR proteins. The proposed NarL site was found in a perfect-symmetry element. The aeg-46.5 regulatory elements are adjacent to, but divergent from, those of the eco gene.

Molecular characterization of the Escherichia coli htrD gene: cloning, sequence, regulation, and involvement with cytochrome d oxidase

The Escherichia coli htrD gene was originally isolated during a search for new genes required for growth at high temperature. Insertional inactivation of htrD leads to a pleiotropic phenotype characterized by temperature-sensitive growth in rich medium, H2O2 sensitivity, and sensitivity to cysteine. The htrD gene was cloned and sequenced, and an htrD::mini-Tn10 insertion mutation was mapped within this gene. The htrD gene was shown to encode a protein of approximately 17.5 kDa. Expression of the htrD gene was examined by using an phi (htrD-lacZ) operon fusion. It was found that htrD is not temperature regulated and therefore is not a heat shock gene. Further study revealed that htrD expression is increased under aerobic growth conditions. Conversely, under anaerobic growth conditions, htrD expression is decreased. In addition, a mutation within the nearby cydD gene was found to drastically reduce htrD expression under all conditions tested. These results indicate that htrD is somehow involved in aerobic respiration and that the cydD gene product is necessary for htrD gene expression. In agreement with this conclusion, htrD mutant bacteria are unable to oxidize the cytochrome d-specific electron donor N,N,N',N'-tetramethyl-p-phenylenediamine.

Characterization of a novel regulatory gene aepA that controls extracellular enzyme production in the phytopathogenic bacterium Erwinia carotovora subsp. carotovora.

Erwinia carotovora subsp. carotovora strain Ecc71 produces an array of extracellular enzymes including pectate lyase (Pel), polygalacturonase, cellulase, and protease. In strain Ecc71, these enzymes are coregulated by aepA, which encodes an activator of extracellular protein production (H. Murata, J. L. McEvoy, A. Chatterjee, A. Collmer, and A. K. Chatterjee, Mol. Plant-Microbe Interact, 4:239-246, 1991). The nucleotide sequence of a 2.7-kb aepA+ DNA segment revealed an open reading frame (ORF) of 1,395 bp which matches with the size of the aepA transcript determined by Northern blot analysis. aepA is predicted to encode a protein of 465 amino acid residues with a molecular mass of approximately 51 kDa and a pI of 6.52. The occurrence of a putative signal sequence and several hydrophobic domains suggest membrane localization of AepA. An aepA-lacZ operon fusion was constitutively expressed in E. coli (DH5 alpha) but inducible by pectate and celery extract in E. c. subsp. carotovora (AC5006). These findings suggest that aepA expression may be negatively regulated in E. c. subsp. carotovora. By assaying for the transcript of pel-1, which species a major secreted Pel species in strain Ecc71, and by following the expression of a pel1-lacZ operon fusion we determined that AepA activates pel-1 transcription. The characteristics of aepA including the lack of homology with other prokaryotic regulatory genes indicate that aepA encodes a novel regulatory protein required for extracellular protein production. Whereas homologs of Ecc71 aepA occur in E. c. subsp. carotovora and E. c. subsp. atroseptica strains, activation of exoenzyme production is markedly stimulated by aepA in E. c. subsp. carotovora than in E. c. subsp. atroseptica.

Transport of 5-aminolevulinic acid by the dipeptide permease in Salmonella typhimurium.

In a previous search for mutants of Salmonella typhimurium that are defective in heme synthesis, one class that is apparently defective in 5-aminolevulinic acid (ALA) uptake (alu) was found. Here, I describe the characterization of these mutations. The mutations all map to a single locus near 77.5 min on the genetic map, which is transcribed counterclockwise. Nutritional tests, genetic and physical mapping, and partial DNA sequence analysis revealed that alu mutants are defective in a periplasmic binding protein-dependent permease that also transports dipeptides, encoded by the dpp operon. The uptake of labeled ALA is defective in dpp mutants and is markedly increased in a strain that has elevated transcription of the dpp locus. Unlabeled L-leucyl-glycine competes with labeled ALA for uptake. In a strain carrying both a dpp-lac operon fusion and a functional copy of the dpp locus, the expression of beta-galactosidase is not induced by ALA, nor, in a hemL mutant, does expression of dpp change substantially during starvation for ALA. The dipeptide permease displays a relaxed substrate specificity that allows transport of the important nonpeptide nutrient ALA, whose structure is closely related to that of glycyl-glycine.

Requirement of homologous recombination functions for viability of the Escherichia coli cell that lacks RNase HI and exonuclease V activities

rnhA224 and rnhA339::cat mutants which lack RNase HI activity were found to constitutively express the sfiaA::lacZ operon fusion in a recA + lexA +-dependent manner. The sfiA::lacZ expression (indicating SOS induction) in rnhA mutants was increased to higher levels by the introduction of the recD1903 or recB21 mutation. The SOS induction in these cells was further enhanced by nutritional shift up from casamino acid medium to Luria broth. Although the extent by which the recD and recB mutations increased the sfiA expression in rnhA mutants was similar, the rnhA224 recB21 double mutant had plating efficiencies that were 25-fold lower on casamino acid plates and 5 × 10 5-fold lower of Luria broth plates than the respective plating efficiencies of either rnhA224 recD or rnhA::cat recD double mutants. Whereas the recD mutation inactivates the exonuclease activity of the RecBCD (Exo V) enzyme without reducing the recombination proficiency of the mutant, the recB21 mutation abolishes both the exonuclease activity and recombination capability. Therefore, in the absence of both RNase HI and Exo V activities, homologous recombination functions become crucial for viability, particularly in Luria broth. Introduction of mutations in recA, recJ and recN exacerbated the phenotypes. It is proposed that R-loops which persist due to the lack of RNase HI activity can be removed by two alternative routes of DNA repair: one involving Exo V, Exo I and DNA polymerase I, and the other involving both the RecBCD and RecF pathways of homologous recombination activities. The isolation of RNA polymerase mutants that constitutively express the SOS response at high levels and exhibit remarkable broth-sensitivity lend strong support to the concention that increased amounts of the persisting R-loop in rnhA mutants growing in Luria broth give rise to a stronger SOS response.

Regulation of narK gene expression in Escherichia coli in response to anaerobiosis, nitrate, iron, and molybdenum.

The regulation of the narK gene in Escherichia coli was studied by constructing narK-lacZ gene and operon fusions and analyzing their expression in various mutant strains in response to changes in cell growth conditions. Expression of narK-lacZ was induced 110-fold by a shift to anaerobic growth and a further 8-fold by the presence of nitrate. The fnr gene product mediates this anaerobic response, while nitrate control is mediated by the narL, narX, and narQ gene products. The narX and narQ gene products were shown to sense nitrate independently of one another and could each activate narK expression in a NarL-dependent manner. We provide the first evidence that NarL and FNR interact to ensure optimal expression of narK. IHF and Fis proteins are also required for full activation of narK expression, and their roles in DNA bending are discussed. Finally, the availability of molybdate and iron ions is necessary for optimal narK expression, whereas the availability of nitrite is not. Although the role of the narK gene product in cell metabolism remains uncertain, the pattern of narK gene expression is consistent with a proposed role of NarK in nitrate uptake by the cell for nitrate-linked electron transport.

Two-stage control of an oxidative stress regulon: the Escherichia coli SoxR protein triggers redox-inducible expression of the soxS regulatory gene.

Escherichia coli responds to the redox stress imposed by superoxide-generating agents such as paraquat by activating the synthesis of as many as 80 polypeptides. Expression of a key group of these inducible proteins is controlled at the transcriptional level by the soxRS locus (the soxRS regulon). A two-stage control system was hypothesized for soxRS, in which an intracellular redox signal would trigger the SoxR protein as a transcriptional activator of the soxS gene and the resulting increased levels of SoxS protein would activate transcription of the various soxRS regulon genes (B. Demple and C.F. Amábile Cuevas, Cell 67:837-839, 1990). We have constructed operon fusions of the E. coli lac genes to the soxS promoter to monitor soxS transcription. Expression from the soxS promoter is strongly inducible by paraquat in a manner strictly dependent on a functional soxR gene. Several other superoxide-generating agents also trigger soxR(+)-dependent soxS expression, and the inductions by paraquat and phenazine methosulfate were dependent on the presence of oxygen. Numerous other oxidative stress agents (H2O2, gamma rays, heat shock, etc.) failed to induce soxS, while aerobic growth of superoxide dismutase-deficient bacteria triggered soxR-dependent soxS expression. These results indicate a specific redox signal for soxS induction. A direct role for SoxR protein in the activation of the soxS gene is indicated by band-shift and DNase I footprinting experiments that demonstrate specific binding of the SoxR protein in cell extracts to the soxS promoter. The mode of SoxR binding to DNA appears to be similar to that of its homolog MerR in that the SoxR footprint spans the -10 to -35 region of the soxS promoter.

The DNA supercoiling-sensitive expression of the Salmonella typhimurium his operon requires the his attenuator and is modulated by anaerobiosis and by osmolarity.

Bacterial cells possess a subset of genes whose expression correlates with changes in DNA supercoiling brought about by anaerobic growth and by growth at high osmolarity. It has been shown previously that expression of the histidine biosynthetic operon of Salmonella typhimurium is derepressed by relaxation of supercoiled DNA. Here, we confirm that a his::MudJ operon fusion in S. typhimurium can be induced by treatment with the DNA gyrase inhibitor novobiocin in a dose-dependent manner, and show that the level of derepression is higher in stationary phase than in mid-exponential phase cultures. Furthermore, expression of his is repressed by anaerobiosis and by osmolarity, two environmental parameters which increase the negative supercoiling of bacterial DNA. Novobiocin induction of his is also repressed by growing the cells either at high osmolarity or anaerobically. Both environmental repression and novobiocin induction of his require the his attenuator. In addition, derepression of his expression by novobiocin and its repression by anaerobiosis or osmolarity are independent of the stringent response gene, relA.

Anaerobic control of colicin E1 production.

Expression of the cea gene, which is carried by the ColE1 plasmid and which encodes colicin E1, was found to be greatly increased when the cells were grown anaerobically. By using cea-lacZ fusions to quantitate expression, aerobic levels were found to be only a few percent of the anaerobic levels. The anaerobic increase in expression was observed both in protein and in operon fusions, indicating that its regulation occurred at the level of transcription. It was also found to require a functional fnr gene and to occur when the cea-lacZ fusion was present as a single copy in the bacterial chromosome instead of in the multicopy ColE1 plasmid. Anaerobic expression was regulated by the SOS response and catabolite repression as is aerobic expression. The start site of the mRNA produced under anaerobic conditions was mapped by primer extension and found to be the same as the start for mRNA produced under aerobic conditions. These observations show that the cea gene is anaerobically regulated and that the Fnr protein is a positive regulator of transcription of this gene.

Oral immunization against cholera toxin with a live Yersinia enterocolitica carrier in mice

The 70-kb pYV plasmid of Yersinia enterocolitica directs the synthesis and secretion of several virulence determinants called Yops. These proteins are produced during the invasion of the host tissues and induce a strong antibody response. The yop genes are transcribed from strong promoters activated by a common transcription activator. Recombinant Y. enterocolitica strains expressing the B subunit of the cholera toxin were constructed from a yopH-ctxB operon fusion. Integration of the gene ctxB in the pYV plasmid itself, by a double crossing over, ensured its stability in the infecting bacteria. Oral inoculation of recombinant bacteria in mice elicited serum and intestinal antibody responses and resulted in protection of the immunized mice against a cholera toxin challenge. Secretory immunoglobulin A antibodies against the cholera toxin B subunit occurred not only in the intestines but also in the respiratory tract.

Genetic evidence for an activator required for induction of pectin lyase in Erwinia carotovora subsp. carotovora by DNA-damaging agents.

In Erwinia carotovora subsp. carotovora 71, the induction of pectin lyase (Pnl), the bacteriocin carotovoricin (Ctv), and cellular lysis (Lss) requires a RecA function. We obtained mutants wherein a pleiotropic defect, i.e., the lack of induction with mitomycin C, is not restored by the recA+ DNA. From a genomic library of strain 71, a cosmid (pAKC280) that restored induction of Pnl, Ctv, and Lss by mitomycin C was isolated. The activator function, designated Rdg for regulator of damage-inducible genes, was localized by subcloning and insertional mutagenesis to a 2.6-kb region within a 6.7-kb EcoRI fragment. An rdg-lacZ operon fusion was inducible by mitomycin C in RecA+ but not RecA- derivatives of E. carotovora subsp. carotovora 71 and Escherichia coli. A RecA+ E. coli strain carrying only a PnlA+ plasmid was not inducible for Pnl production; however, when both a PnlA+ plasmid and a Rdg+ plasmid were present, the transcription of pnlA and the production of the enzyme were activated by mitomycin C. The size of the pnlA transcript produced in E. coli was identical to that of the transcript produced by E. carotovora subsp. carotovora 71, suggesting that the same promoter and termination sequences were being utilized in these bacteria.

δ-Aminolevulinate couples cycA transcription to changes in heme availability in Rhodobacter sphaeroides

In this paper, the response of the transcriptional control region of the Rhodobacter sphaeroides cytochrome c 2 gene, cycA, to intermediates in heme biosynthesis was studied. To determine if cycA transcription was regulated by heme availability, several precursors or analogs of tetrapyrroles were tested. Addition of δ-aminolevulinate (ALA), the first committed intermediate in heme biosynthesis, was shown to inhibit cycA transcription initiation at both the upstream and downstream promoter regions. In addition, an ALA auxotroph, which can grow in the presence of high levels of ALA, showed a 5 to 7-fold reduction in steady-state transcription from cycA::lacZYA operon fusions. To identify genetic elements responsible for negative regulation by ALA, trans-acting mutants with increased expression of cycA were isolated that were resistant to growth inhibition by the heme analog cohemin. These cohemin-resistant mutants (Chr) have elevated levels of several cycA transcripts and they contain cycA transcripts that had not previously been detected in wild-type cells. In addition, cycA transcription in the Chr mutants continues after the addition of ALA. Finally, we found that Chr mutants have increased ALA synthase activity, suggesting that synthesis of cytochrome c 2 and ALA synthase are controlled by a common gene product whose activity has been modified in these mutants. A model is presented to explain how changes in tetrapyrrole intermediates could provide an effective signal to control both cycA transcription and ALA synthase synthesis in R. sphaeroides.

Use of glucose starvation to limit growth and induce protein production in Escherichia coli

The use of glucose starvation to uncouple the production of recombinant beta-galactosidase from cell growth in Escherichia coli was investigated. A lacZ operon fusion to the carbon starvation-inducible cst-1 locus was used to control beta-galactosidase synthesis. beta-Galactosidase induction was observed only under aerobic starvation conditions, and its expression continued for 6 h following the onset of glucose starvation. The cessation of beta-galactosidase expression closely correlated with the exhaustion of acetate, an overflow metabolite of glucose, from the culture medium. Our results suggest the primary role of acetate in cst-1-controlled protein expression is that of an energy source. Using this information, we metered acetate to a glucose-starved culture and produced a metabolically sluggish state, where growth was limited to a low linear rate and production of recombinant beta-galactosidase occurred continuously throughout the experiment. The cst-1 controlled beta-galactosidase synthesis was also induced at low dilution rates in a glucose-limited chemostat, suggesting possible applications to high-density cell systems such as glucose-limited recycle reactors. This work demonstrates that by using an appropriate promoter system and nutrient limitation, growth can be restrained while recombinant protein production is induced and maintained.

Leucine-responsive regulatory protein controls the expression of both the pap and fan pili operons in Escherichia coli.

The methylation blocking factor gene (mbf) in Escherichia coli is required for specific methylation inhibition of two DNA GATC sites upstream of the papBA pilin promoter and transcriptional activation of pap. Complementation and mutational analysis using pap-lac and ilvIH-lac operon fusions indicates that the mbf gene is identical to a recently described global regulatory gene lrp (leucine-responsive regulatory protein) that acts as a positive regulator of some genes and a negative regulator of others in E. coli. DNA sequence analysis of an mbf::mTn10 insertion showed that the mbfDNA sequence was identical to lrp. Thus Lrp inhibits DNA methylation at specific GATC sites. We also show that Lrp positively regulates transcription of the fan operon, which encodes K99 pili of diarrheagenic E. coli. Purified Lrp was found to bind to DNA fragments encompassing the pap and fan promoters, which is consistent with previous results indicating that Lrp controls gene expression by binding to regulatory DNA sites. Exogenous leucine significantly reduced fan transcription and K99 pili expression, similar to results obtained with the ilvIH operon. However, pap gene expression was unresponsive to leucine, which distinguishes pap from other lrp-regulated genes whose expression is modulated by leucine.

Transcriptional and maturational effects of manganese and iron on the biosynthesis of manganese-superoxide dismutase in Escherichia coli.

Anaerobically grown Escherichia coli contain an enzymatically active iron superoxide dismutase (Fe2-FeSOD) and an inactive iron-substituted manganese superoxide dismutase (Fe2-MnSOD). The anaerobic electron sink, nitrate plus paraquat, enhanced biosynthesis of the MnSOD polypeptide, with accumulation of inactive Fe2-MnSOD. The oxidant, diamide, in contrast, allowed anaerobic production of the active forms of MnSOD, i.e. Mn2-MnSOD and Mn/Fe-MnSOD. Nutritional supplementation with Mn(II) favored occupancy of the MnSOD active site with manganese and allowed anaerobic accumulation of Mn2-MnSOD in the absence of diamide. Enrichment of the anaerobic growth medium with Fe(II) both suppressed biosynthesis of the MnSOD polypeptide and inhibited formation of the active manganese-containing forms. A tac-sodA operon fusion was used to examine the effects of chelating agents and metals on maturation of nascent MnSOD, independent from the transcriptional effects these agents impose. Isopropyl-1-thio-beta-D-galactopyranoside (IPTG) elicited anaerobic biosynthesis of MnSOD, which accumulated as the inactive Fe2-MnSOD. Diamide, with IPTG, allowed formation of active Mn/Fe-MnSOD while 1,10-phenanthroline with IPTG resulted in accumulation of Mn2-MnSOD. These results suggest that iron participates in the redox-sensitive control of the formation of active MnSOD at two levels, i.e. that of transcription as well as that of maturation. During maturation of the nascent MnSOD polypeptide, iron and manganese compete for the metal-binding site; anaerobic conditions favor iron-binding, whereas oxidants, such as dioxygen or diamide, favor binding of manganese.