ArcA and AppY antagonize IscR repression of hydrogenase-1 expression under anaerobic conditions, revealing a novel mode of O2 regulation of gene expression in Escherichia coli.

Transcription of the Escherichia coli hydrogenase-1 operon (hyaABCDEF) is increased by the transcription factors ArcA and AppY under anaerobic growth conditions. However, IscR, which represses transcription of the hyaA promoter (P(hyaA)) under aerobic conditions, was not known to repress transcription of this promoter under anaerobic conditions. Here, we report that ArcA and AppY increase P(hyaA) expression under anaerobic conditions by antagonizing IscR binding at P(hyaA), since IscR repression is observed when either ArcA or AppY is eliminated. The ability of ArcA and AppY to act as antirepressors of IscR repression of P(hyaA) depended on IscR levels, suggesting that IscR competes with ArcA and/or AppY for binding. In support of this competition model, electrophoretic mobility shift assays and DNase I footprinting showed that the ArcA and IscR binding sites overlap and that binding of ArcA and IscR is mutually exclusive. Unexpectedly, IscR with a C92A mutation (IscR-C92A), which mimics the clusterless form of the protein that is present predominantly under aerobic conditions, was a better repressor under anaerobic conditions of both P(hyaA) and a constitutive promoter containing the IscR binding site from P(hyaA) than wild-type IscR, which is predominantly in the [2Fe-2S] form under anaerobic conditions. This observation could not be explained by differences in DNA binding affinities or IscR levels, so we conclude that [2Fe-2S]-IscR is a weaker repressor of P(hyaA) than clusterless IscR. In sum, a combination of ArcA and AppY antirepression of IscR function, lower levels of IscR, and weak repression by [2Fe-2S]-IscR leads to increased P(hyaA) expression under anaerobic conditions.

Sequence-specific binding to a subset of IscR-regulated promoters does not require IscR Fe-S cluster ligation.

IscR is an Fe-S protein that functions as a transcriptional regulator of Fe-S biogenesis and other Fe-S protein-encoding genes in Escherichia coli. In this study, we investigated the requirement for the ligation of the [2Fe-2S] cluster of IscR to regulate a subset of IscR target promoters (P(hyaA), P(ydiU), P(napF), and P(hybO)) and defined the requirements for sequence-specific binding to the IscR target site in the hyaA promoter region. In contrast to previous results with the iscR promoter, we found that the Fe-S cluster is dispensable for IscR regulation of P(hyaA), P(ydiU), P(napF), and P(hybO), since IscR mutants containing alanine substitutions of the cysteine Fe-S ligands retained IscR-dependent regulation of these promoters in vivo. In vitro assays showed that both [2Fe-2S]-IscR and an IscR mutant lacking the cluster (IscR-C92A/C98A/C104A) bound the hya site with similar affinity, explaining why the mutant protein retained its ability to repress P(hyaA) in vivo. Characterization of the oligomeric state of IscR showed that both apo-IscR and [2Fe-2S]-IscR were dimers in solution, and four protomers of either form bound to the hya site. Also, binding of either apo- or [2Fe-2S]-IscR to the hya site showed cooperativity, suggesting that both forms interact similarly with the target site. Analysis of mutations in the hya site using DNA competition assays showed that apo-IscR most likely recognizes an imperfect palindrome within the hya promoter. Furthermore, the strength of apo-IscR binding to P(sufA), P(ydiU), P(napF), and P(hybO) IscR sites correlated with the number of matches to the hya site bases shown to be important in the competition assay. Thus, our data indicated that, unexpectedly, apo-IscR is a site-specific DNA-binding protein, and the role of apo-IscR needs to be considered in developing models for how IscR globally regulates transcription.

IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins.

IscR (iron-sulfur cluster regulator) is encoded by an ORF located immediately upstream of genes coding for the Escherichia coli Fe-S cluster assembly proteins, IscS, IscU, and IscA. IscR shares amino acid similarity with MarA, a member of the MarA/SoxS/Rob family of transcription factors. In this study, we found that IscR functions as a repressor of the iscRSUA operon, because strains deleted for iscR have increased expression of this operon. In addition, in vitro transcription reactions established a direct role for IscR in repression of the iscR promoter. Analysis of IscR by electron paramagnetic resonance showed that the anaerobically isolated protein contains a [2Fe-2S](1+) cluster. The Fe-S cluster appears to be important for IscR function, because repression of iscR expression is significantly reduced in strains containing null mutations of the Fe-S cluster assembly genes iscS or hscA. The finding that IscR activity is decreased in strain backgrounds in which Fe-S cluster assembly is impaired suggests that this protein may be part of a novel autoregulatory mechanism that senses the Fe-S cluster assembly status of cells.

Characterization of the dimerization domain in the FNR transcription factor.

The global anaerobic regulator FNR from Escherichia coli is a dimeric Fe-S protein that is inactivated by O(2) through disruption of its [4Fe-4S] cluster and conversion to a monomeric form. As a first step in elucidating the molecular interactions that control FNR dimerization, we have performed alanine-scanning mutagenesis of a potential dimerization domain. Replacement of many hydrophobic residues (Met-143, Met-144, Leu-146, Met-147, Ile-151, Met-157, and Ile-158) and two charged residues (Arg-140 and Arg-145) with Ala decreased FNR activity in vivo. Size exclusion chromatography and Fe-S cluster analysis of three representative mutant proteins, FNR-M147A, FNR-I151A, and FNR-I158A, showed that the Ala substitutions produced specific defects in dimerization. Because hydrophobic side chains are known to stabilize subunit-subunit interactions between alpha-helices, we propose that Met-147, Ile-151, and Ile-158 lie on the same face of an alpha-helix that constitutes a dimerization interface. This alignment would also position Arg-140, Met-144, and Asp-154 on the same helical face. In support of the unusual positioning of a negatively charged residue at the dimer interface, we found that replacing Asp-154 with Ala repaired the defects caused by Ala substitutions of other residues located on the same helical face. These data also suggest that Asp-154 has an inhibitory effect on dimerization, which may be a key element in the control of FNR dimerization by O(2) availability.

FNR-dependent activation of the class II dmsA and narG promoters of Escherichia coli requires FNR-activating regions 1 and 3.

In Escherichia coli, the anaerobic expression of genes encoding the nitrate (narGHJI) and dimethyl sulphoxide (dmsABC) terminal reductases is stimulated by the global anaerobic regulator FNR. The ability of FNR to activate transcription initiation has been proposed to be dependent on protein-protein interactions between RNA polymerase and two activating regions (AR) of FNR, FNR-AR1 and FNR-AR3. To further our understanding of the role of FNR-AR1 and FNR-AR3 in transcription activation, we measured the effects of FNR-AR mutants on expression of the narG and dmsA promoters, PnarG and PdmsA. All the FNR-AR1 (FNR-S73F, FNR-T118A, FNR-S187P), FNR-AR3 (FNR-G85A) and FNR-AR1-AR3 (FNR-G85A-S187P) mutants that were tested decreased expression from PnarG and PdmsA in vivo. Transcription assays of PdmsA also showed that the FNR-AR mutant proteins impaired transcription activation in vitro. Furthermore, DNase I footprinting analysis confirmed that this transcription defect was not a result of altered DNA-binding properties. The function of FNR-S187P and FNR-G85A was also measured in strains containing sigma70 mutants (sigma70-K593A, sigma70-R596A and sigma70-K597A) known to be impaired in FNR-dependent transcription activation. Of all of the combinations analysed, only FNR-G85 and sigma70-K597 showed a genetic interaction, supporting the notion that FNR-AR3 and sigma70 interact functionally in the process of transcription activation. Lastly, the transcription activation defect of the FNR-AR1 and FNR-AR3 mutants was greatly reduced when expression of PnarG was assayed in the presence of nitrate. As these growth conditions promote maximal activity of PnarG as a result of the combined function of NarL, IHF and FNR, these results suggest that the requirements for FNR-AR1 and FNR-AR3 are altered in the presence of additional activators.

The cysteine desulfurase, IscS, has a major role in in vivo Fe-S cluster formation in Escherichia coli.

The cysteine desulfurase, IscS, provides sulfur for Fe-S cluster synthesis in vitro, but a role for IscS in in vivo Fe-S cluster formation has yet to be established. To study the in vivo function of IscS in Escherichia coli, a strain lacking IscS was constructed and characterized. Using this iscS deletion strain, we have observed decreased specific activities for proteins containing [4Fe-4S] clusters from soluble (aconitase B, 6-phosphogluconate dehydratase, glutamate synthase, fumarase A, and FNR) and membrane-bound proteins (NADH dehydrogenase I and succinate dehydrogenase). A specific role for IscS in in vivo Fe-S cluster assembly was demonstrated by showing that an Fe-S cluster independent mutant of FNR is unaffected by the lack of IscS. These data support the conclusion that, via its cysteine desulfurase activity, IscS provides the sulfur that subsequently becomes incorporated during in vivo Fe-S cluster synthesis. We also have characterized a growth phenotype associated with the loss of IscS. Under aerobic conditions the deletion of IscS caused an auxotrophy for thiamine and nicotinic acid, whereas under anaerobic conditions, only nicotinic acid was required. The lack of IscS also had a general effect on the growth of E. coli because the iscS deletion strain grew at half the rate of wild type in many types of media even when the auxotrophies were satisfied.

Substitution of leucine 28 with histidine in the Escherichia coli transcription factor FNR results in increased stability of the [4Fe-4S](2+) cluster to oxygen.

To understand the role of the [4Fe-4S](2+) cluster in controlling the activity of the Escherichia coli transcription factor FNR (fumarate nitrate reduction) during changes in O(2) availability, we have characterized a mutant FNR protein containing a substitution of Leu-28 with His (FNR-L28H) which, unlike its wild type (WT) counterpart, is functional under aerobic growth conditions. The His-28 substitution appears to stabilize the [4Fe-4S](2+) cluster of FNR-L28H in the presence of O(2) because air-exposed FNR-L28H did not undergo the rapid [4Fe-4S](2+) to [2Fe-2S](2+) cluster conversion or concomitant loss in site-specific DNA binding and dimerization, which are characteristic of WT-FNR under these conditions. This increased cluster stability was not a result of His-28 replacing the WT-FNR cluster ligands because substitution of any of these four Cys residues (cysteine 20, 23, 29, or 122) with Ser resulted in [4Fe-4S](2+) cluster-deficient preparations of FNR-L28H. The Mössbauer spectra of FNR-L28H indicated that the coordination environment of the [4Fe-4S](2+) cluster did not differ from that of WT-FNR. Whole cell Mössbauer spectroscopy showed that aerobically grown cells overexpressing FNR-L28H had levels of the FNR species containing the [4Fe-4S](2+) cluster similar to those of cells grown under anaerobic conditions. Thus, the increase in cluster stability is sufficient to allow accumulation of the [4Fe-4S](2+) cluster form of FNR-L28H under aerobic conditions and provides a reasonable explanation for why this mutant protein is functional under aerobic growth conditions. From these results, we present a model to explain how WT-FNR is normally inactivated under aerobic growth conditions.

Fe-S proteins in sensing and regulatory functions.

In the past five to ten years, it has become increasingly apparent that the function of Fe-S clusters is not limited to electron transfer, a function implicit in their discovery. We now know that the vulnerability of these structures to oxidative destruction is used by nature in sensing O2, iron, and possibly also nitric oxide. Changes in the oxidation state of Fe-S clusters can also serve as a reversible switch.

Mössbauer spectroscopy as a tool for the study of activation/inactivation of the transcription regulator FNR in whole cells of Escherichia coli.

The global regulator FNR (for fumarate nitrate reduction) controls the transcription of >100 genes whose products facilitate adaptation of Escherichia coli to growth under O2-limiting conditions. Previous Mössbauer studies have shown that anaerobically purified FNR contains a [4Fe-4S]2+ cluster that, on exposure to oxygen, is converted into a [2Fe-2S]2+ cluster, a process that decreases DNA binding by FNR. Using 57Fe Mössbauer spectroscopy of E. coli cells containing overexpressed FNR, we show here that the same cluster conversion also occurs in vivo on exposure to O2. Furthermore, the data show that a significant amount of the [4Fe-4S]2+ cluster is regenerated when the cells are shifted back to an anaerobic environment. The present study also demonstrates that 57Fe Mössbauer spectroscopy can be employed to study the in vivo behavior of (overexpressed) proteins. The use of this technique to study other iron-containing cell components is discussed.

Fnr, NarP, and NarL regulation of Escherichia coli K-12 napF (periplasmic nitrate reductase) operon transcription in vitro.

The expression of several Escherichia coli operons is activated by the Fnr protein during anaerobic growth and is further controlled in response to nitrate and nitrite by the homologous response regulators, NarL and NarP. Among these operons, the napF operon, encoding a periplasmic nitrate reductase, has unique features with respect to its Fnr-, NarL-, and NarP-dependent regulation. First, the Fnr-binding site is unusually located compared to the control regions of most other Fnr-activated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation. Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein. In this study, we used DNase I footprint analysis to confirm our previous assignment of the unusual location of the Fnr-binding site in the napF control region. In addition, the in vivo effects of Fnr-positive control mutations on napF operon expression indicate that the napF promoter is atypical with respect to Fnr-mediated activation. The transcriptional regulation of napF was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP proteins. These in vitro transcription experiments demonstrate that, in the presence of Fnr, the NarP protein causes efficient transcription activation whereas the NarL protein does not. This suggests that Fnr and NarP may act synergistically to activate napF operon expression. As observed in vivo, this activation by Fnr and NarP is antagonized by the addition of NarL in vitro.

Oxygen sensing by the global regulator, FNR: the role of the iron-sulfur cluster.

FNR is a global regulator that controls transcription of genes whose functions facilitate adaptation to growth under O2 limiting conditions. It has long been appreciated that the activity of FNR must be regulated by O2 availability, since FNR dependent gene expression is observed in vivo only under anaerobic conditions, while similar levels of this protein are present in both aerobic and anaerobic grown cells. Recent progress in this field has shown that anaerobically purified FNR contains a [4Fe-4S]2+ cluster and that this [4Fe-4S]2+ cluster is sufficiently unstable toward O2 to make it suitable as an O2 sensor. The presence of the [4Fe-4S] cluster increases dimerization of FNR which is correlated with an increase in site-specific DNA binding of FNR, a property expected of transcription factors of the FNR/CRP family. According to Mössbauer spectroscopy on purified FNR and cells containing overexpressed FNR, the [4Fe-4S]2+ cluster of FNR is converted by O2 to a [2Fe-2S]2+ in high yield. The [2Fe-2S]2+ cluster can be reconverted to the [4Fe-4S]2+ cluster on reduction with dithionite in vitro raising the possibility that the [2Fe-2S]2+ cluster is a biologically inactive intermediate which may be more readily available for reconstitution into the [4Fe-4S]2+ form than the Fe-free apoform. The ability to observe, by Mössbauer spectroscopy, the Fe-S clusters of FNR in cells containing high levels of FNR should be of value in further unraveling how FNR functions in vivo. Attempts to reduce the [4Fe-4S]2+ cluster of FNR with dithionite indicated that the redox potential of the +1/+2 couple is < or = -650 mV and that the [4Fe-4S]+ cluster form is, therefore, not likely to occur in vivo.

Iron-sulfur cluster disassembly in the FNR protein of Escherichia coli by O2: [4Fe-4S] to [2Fe-2S] conversion with loss of biological activity.

The transcription factor FNR (fumarate nitrate reduction) requires the presence of an iron-sulfur (Fe-S) cluster for its function as a global transcription regulator in Escherichia coli when oxygen becomes scarce. To define the oxidation state and type of Fe-S cluster present in the active form of FNR, we have studied anaerobically purified FNR with Mössbauer spectroscopy. Our data showed that this form of FNR contained a [4Fe-4S]2+ cluster (delta = 0.45 mm/s; DeltaEQ = 1.22 mm/s) and that the [4Fe-4S]2+ cluster was rapidly destroyed on exposure of FNR to air. Under these conditions, the yellow-green active form of FNR turned deep red; analysis of sulfide indicated that 70% of the labile sulfide was still present, suggesting that the Fe-S cluster had been converted into a different form. Little [3Fe-4S] cluster was, however, detected by EPR. According to Mössbauer spectroscopy, the [4Fe-4S]2+ cluster was converted in about 60% yield to a [2Fe-2S]2+ cluster (delta = 0.28 mm/s; DeltaEQ = 0.58 mm/s) following 17 min of exposure to air. The [2Fe-2S]2+ cluster form of FNR was much more stable to oxygen, but was unable to sustain biological activity (e.g., DNA binding). However, DNA binding and the absorption spectrum characteristic of the [4Fe-4S]2+ cluster could be largely restored from the [2Fe-2S]2+ form when Cys, Fe, DTT, and the NifS protein were added. It has yet to be determined whether the form of FNR containing the [2Fe-2S]2+ cluster has any biological significance, e.g., as an in vivo intermediate that is more rapidly converted to the active form than the apoprotein.

DNA binding and dimerization of the Fe-S-containing FNR protein from Escherichia coli are regulated by oxygen.

The transcription factor FNR from Escherichia coli regulates transcription of genes in response to oxygen deprivation. To determine how the activity of FNR is regulated by oxygen, a form of FNR had to be isolated that had properties similar to those observed in vivo. This was accomplished by purification of an FNR fraction which exhibited enhanced DNA binding in the absence of oxygen. Iron and sulfide analyses of this FNR fraction indicated the presence of an Fe-S cluster. To determine the type of Fe-S cluster present, an oxygen-stable mutant protein LH28-DA154 was also analyzed since FNR LH28-DA154 purified anoxically contained almost 3-fold more iron and sulfide than the wild-type protein. Based on the sulfide analysis, the stoichiometry (3.3 mol of S2-/FNR monomer) was consistent with either one [4Fe-4S] or two [2Fe-2S] clusters per mutant FNR monomer. However, since FNR has only four Cys residues as potential cluster ligands and an EPR signal typical of a 3Fe-4S cluster was detected on oxidation, we conclude that there is one [4Fe-4S] cluster present per monomer of FNR LH28-DA154. We assume that the wild type also contains one [4Fe-4S] cluster per monomer and that the lower amounts of iron and sulfide observed per monomer were due to partial occupancy. Consistent with this, the Fe-S cluster in the wild-type protein was found to be extremely oxygen-labile. In addition, molecular-sieve chromatographic analysis showed that the majority of the anoxically purified protein was a dimer as compared to aerobically purified FNR which is a monomer. The loss of the Fe-S cluster by exposure to oxygen was associated with a conversion to the monomeric form and decreased DNA binding. Taken together, these observations suggest that oxygen regulates the activity of wild-type FNR through the lability of the Fe-S cluster to oxygen.

Characterization of FNR* mutant proteins indicates two distinct mechanisms for altering oxygen regulation of the Escherichia coli transcription factor FNR.

In order to gain insight into the mechanism by which the Escherichia coli transcription factor FNR* is activated in response to anaerobiosis, we have analyzed FNR mutant proteins which, unlike the wild-type protein, stimulate gene expression in the presence of oxygen in vivo. Cell extracts containing seven different FNR* mutant proteins were tested in vitro for the ability to bind to the FNR consensus DNA site in a gel retardation assay under aerobic conditions. At the concentration of protein tested, only extracts which contained FNR* mutant proteins with amino acid substitutions at position 154 showed significant DNA binding. The three position-154 FNR* mutant proteins could be further distinguished from the other mutant proteins by analysis of the in vivo phenotypes of FNR* proteins containing amino acid substitutions at either of two essential cysteine residues. In the presence of oxygen, FNR* mutant proteins with amino acid substitutions at position 154 were the least affected when either Cys-23 or Cys-122 was substituted for Ser. On the basis of these in vivo and in vitro analyses, FNR* mutant proteins appear to segregate into at least two classes. Thus, it appears that each class of FNR* substitutions alters the normal pathway of FNR activation in response to oxygen deprivation by a different mechanism.

Association of a polynuclear iron-sulfur center with a mutant FNR protein enhances DNA binding.

In the facultative anaerobe Escherichia coli, the transcription factor FNR (fumarate nitrate reduction) regulates gene expression in response to oxygen deprivation. To investigate how the activity of FNR is regulated by oxygen availability, two mutant proteins, DA154 and LH28-DA154, which have enhanced in vivo activity in the presence of oxygen, were purified and compared. Unlike other previously examined FNR preparations, the absorption spectrum of LH28-DA154 had two maxima at 324 nm and 419 nm, typical of iron-sulfur (Fe-S)-containing proteins. Consistent with these data, metal analysis showed that only the LH28-DA154 protein contained a significant amount of iron and acid-labile sulfide, and, by low temperature EPR spectroscopy, a signal typical of a [3Fe-4S]+ cluster was detected. The LH28-DA154 protein that contained the Fe-S cluster also contained a higher proportion of dimers and had a 3- to 4-fold higher apparent affinity for the target DNA than the DA154 protein. In agreement with this, we found that when the LH28-DA154 protein was treated with an iron chelator (alpha,alpha'-dipyridyl), it lost its characteristic absorption and the apparent affinity for DNA was reduced 6-fold. However, increased DNA binding and the characteristic absorption spectrum could be restored by in vitro reconstitution of the Fe-S center. DNA binding of the LH28-DA154 protein was also affected by the redox state of the Fe-S center, since protein exposed to oxygen bound 1/10th as much DNA as the protein reduced anaerobically with dithionite. The observation that DNA binding is enhanced when the Fe-S center is reduced indicates that the redox state of the Fe-S center affects the DNA-binding activity of this protein and suggests a possible mechanism for regulation of the wild-type protein.

In vitro analysis of a constitutively active mutant form of the Escherichia coli global transcription factor FNR.

The Escherichia coli transcription factor FNR regulates expression of genes required for the metabolic switch between aerobic and anaerobic respiration. In order to investigate how FNR controls transcription of its target operons, DNA binding was examined for both the wild-type (WT) FNR protein and an altered function FNR* protein (DA154) that exhibits enhanced activity in the presence of oxygen both in vivo and in vitro, apparently due to the fact that DA154 is able to dimerize to a greater extent than WT FNR. Electrophoretic mobility shift assays, using a consensus symmetrical FNR target site, revealed that both DA154 and WT FNR gave rise to protein-DNA complexes of indistinguishable electrophoretic mobilities. In addition, an estimate of the molecular weight from the mobility of the DA154-DNA complex indicated that both mutant and WT FNR were dimeric when bound to DNA. Under the same binding conditions, DA154 showed an observed constant of approximately 3 x 10(8) M-1 for the consensus symmetrical target site. In addition, the results of DNA binding competition assays provided evidence that DA154 was a site-specific DNA binding protein, since this mutant protein bound to the consensus symmetrical target site with approximately 40-fold and 250-fold higher affinity than a natural target site from the nar promoter or a non-specific DNA target, respectively. Electrophoretic mobility shift DNA bending assays demonstrated protein-induced DNA bending by both DA154 and WT FNR. In addition, in vitro transcription assays using an FNR-dependent variant of the lac P1 promoter demonstrated levels of transcription activation by DA154 comparable to those observed in vivo. These results provide several new insights into how FNR functions to activate transcription of target genes.

The activity of the Escherichia coli transcription factor FNR is regulated by a change in oligomeric state.

The transcription factor FNR globally regulates gene expression in response to oxygen deprivation in Escherichia coli. To understand how oxygen deprivation activates FNR, a constitutively active FNR* mutant protein, DA154, was studied to determine how this mutant bypassed the normal regulation pathway. When purified from aerobically grown cells, the DA154 protein had a larger apparent native molecular mass and an increased affinity for a consensus FNR target site as compared with wild-type FNR prepared under identical conditions. These results suggested that FNR* DA154 may bypass the normal regulation pathway by converting FNR from an inactive monomer to an active dimer under aerobic conditions. To determine whether wild-type FNR is active as a dimer under anaerobic conditions, FNR mutants were isolated that inhibit the activity of wild-type FNR by forming mixed dimers (i.e., dominant-negative mutants). These dominant-negative FNR mutants were shown to have substitutions in the putative DNA-binding domain and to be defective in binding to a consensus FNR DNA target site in vitro. One representative dominant-negative mutant, EK209, was also shown to be unable to form mixed oligomers in vivo under aerobic conditions, suggesting that FNR may be monomeric in the inactive state. Taken together, these data have led us to propose that under anaerobic conditions FNR is a dimer that is active for DNA binding, and under aerobic conditions, FNR is inactivated by conversion to a monomer.

Fnr mutants that activate gene expression in the presence of oxygen.

The regulatory protein Fnr is required for anaerobic expression of several anaerobic respiratory enzymes in Escherichia coli. To gain insight into how Fnr activity is regulated by oxygen, we have isolated Fnr mutants that increase expression of the nitrate reductase operon in the presence of oxygen (Fnr* mutants). Seven single-amino-acid substitutions that mapped within two regions of Fnr have been characterized. Two mutants mapped adjacent to two Cys residues in the N-terminal Cys cluster. Five Fnr* substitutions mapped to a region of Fnr that is similar to the cyclic AMP-binding domain of the catabolite activator protein (CAP). Within this group, four mutants were clustered in a region analogous to the CAP C helix, which is important in CAP dimer subunit interactions. Taken together, these data implicate regions in Fnr that may be important either in sensing oxygen deprivation or in the conformational change proposed to be necessary for Fnr activation under anaerobic conditions.

Posttranscriptional control of puc operon expression of B800-850 light-harvesting complex formation in Rhodobacter sphaeroides.

The puc operon of Rhodobacter sphaeroides comprises the pucBA structural genes which encode B800-850 light-harvesting beta and alpha polypeptides, respectively. Northern (RNA) blot hybridization analysis of puc operon expression has identified two pucBA-specific transcripts. The small (0.5-kilobase [kb]) transcript encodes the beta and alpha polypeptides and, under photoheterotrophic growth conditions, was approximately 200-fold more abundant than the large (2.3-kb) transcript. The 5' end of the 0.5-kb transcript was mapped at 117 nucleotides upstream from the start of pucB. The 3' ends of the 0.5-kb transcript were mapped to two adjacent nucleotides, which follow a stem-loop structure immediately 3' to the pucA stop codon. Two mutant strains, PUC705-BA and PUC-Pv, were constructed by replacement of the pucBA genes and adjacent DNA in the former case or by insertional interruption of the DNA downstream of the pucBA genes in the latter case. The two mutant strains were devoid of B800-850 complexes during photosynthetic growth but were otherwise apparently normal. The B800-850 phenotype of both PUC705-BA and PUC-Pv was not complemented in trans with a 2.5-kb PstI restriction endonuclease fragment extending from 0.75 kb upstream of pucBA to 1.3 kb downstream of pucBA, despite the presence of the 0.5-kb pucBA-specific transcript. Both of the mutant strains, however, showed restoration of B800-850 expression with a 10.5-kb EcoRI restriction endonuclease fragment in trans encompassing the 2.5-kb PstI fragment. Western immunoblot analysis revealed no B800-850-beta polypeptide as well as no polypeptide designated 15A in either mutant. Nonetheless, under photoheterotrophic growth conditions, the 0.5-kb pucBA-specific transcript was present in PUC-Pv, although no 2.3-kb transcript was detectable. We suggest that the DNA region immediately downstream of pucBA encodes a gene product(s) essential for translational or posttranslational expression of the B800-850 beta and alpha polypeptides.