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FNR Is a transcriptional regulator that controls gene expression in response to oxygen limitation in Escherichia coli. The NADH dehydrogenase II gene (ndh) is repressed by FNR under anaerobic conditions. Repression is not simply due to occlusion of the promoter (-35 and -10) region by FNR because adjacent pairs of FNR monomers were found to bind at two sites centred at -50.5 and -94.5 in the ndh promoter region without preventing RNA polymerase binding. However, contact between RNA polymerase and the -132 to -62 region of the non-coding strand of ndh DNA, and RNA polymerase-mediated open complex formation, were prevented by bound FNR. The upstream FNR-binding site (-94.5) was needed for efficient FNR-dependent repression of ndh transcription in vitro, and also for repression of an ndh-lacZ fusion in vivo. Anaerobic ndh repression may thus involve the binding of two pairs of FNR monomers upstream of the -35 region, which prevents essential RNA polymerase-DNA contacts in the upstream region as well as inhibiting RNA polymerase function by direct FNR interaction. Expression of the ndh-lacZ fusion in an fnr deietion strain was enhanced by anaerobic growth in rich medium or minimal medium supplemented with amino acids. Furthermore, two proteins (Mr 12 000 and 35 000) which interact with and may activate transcription from the ndh promoter under these conditions were detected by gel retardation analysis. These putative amino acid-responsive activators may thus offset FNR-mediated repression and maintain a low level of anaerobic ndh expression for regulating the NAD*NADH ratio during growth in rich media. [ABSTRACT FROM AUTHOR]

Transcript mapping and studies with lacZ translational fusions have shown that the pdhR gene (formerly genA) is the proximal gene of the pdhR-aceE-aceF-lpd operon encoding the pyruvate dehydrogenase (PDH) complex of Escherichia coli. A pdhR∼lpd read-through transcript (7.4 kb) initiating at the pyruvate-inducibte pdh promoter, and a smaller lpd transcript (1.7 kb) initiating at the independent lpd promoter, were identified. Evidence showing that the pdhR gene product negatively regulates the synthesis of the PdhR protein and the PDH complex via the pdh promoter was obtained, with pyruvate (or a derivative) serving as the putative inducing coeffector. The partially purified PdhR protein was also found to specifically retard and protect DNA fragments containing the pdh promoter region. The pdh promoter was not strongly controlled by ArcA, FNR or CRP. [ABSTRACT FROM AUTHOR]

FNR, the activator of anaerobic respiratory genes of Escherichia coli, has previously only been isolated as a protein of Mr 29000, which lacks nine W-terminal amino acid residues. The underlying proteolytic events have been studied with the aim of isolating intact FNR and determining whether cleavage is the result of a physiologically significant intracellular processing mechanism or proteolytic degradation during isolation. The FNR protein was present in aerobically and anaerobically grown bacteria as the intact protein (Mr 30000). Proteolysis only occurred during and shortly after disruption of the bacteria. The production of FNR (M, 29000) must therefore be regarded as an isolation artefact. The proteolysis was caused by a protease which is located outside the cytoplasmic membrane or activated upon disruption of the membrane. Protease Inhibitors directed against serine, cysteine or metallo-proteases failed to prevent cleavage of FNR. In E. coli strain CAG627, proteolysis was greatly reduced making it possible to isolate FNR of Mr 30000. The N-terminal sequence of FNR (Mr 30000) was identical to that predicted from the fnr gene starting with the initiating methionine residue and including a four-cysteine cluster (16)Cys-X3-Cys-X2-Cys--X5-Cys(29). [ABSTRACT FROM AUTHOR]

The ndh gene of Escherichia coli which encodes an NADH dehydrogenase contains a putative FNR-binding site in its upstream non-coding region, and its expression has been investigated using an ndh-lacZ fusion. Expression of the fusion was found to be reduced during anaerobic growth, and experiments with hosts containing an fnr mutation and/or a multicopy fnr+ plasmid indicated that the anaerobic repression of the ndh gene is mediated by the FNR protein. Thus FNR can function as an anaerobic repressor as well as an anaerobic transcriptional activator. The results are consistent with the FNRbinding function attributed to the proposed consensus sequence. Using frdA - and ndh-lacZfusions exhibiting positive and negative regulation by FNR, it was further shown that the depletion of metal ions in growth media with chelating agents mimics oxygen with respect to the activity of FNR. Possible roles for metal ions in the oxygen-sensing pathway associated with FNR function are discussed. [ABSTRACT FROM AUTHOR]

The FNR protein of Escherichia coli is a regulatory protein that activates the transcription of its target genes in response to oxygen limitation. Site-directed mutagenesis was used to show that a 28-residue N-terminal segment containing three cysteines is essential for normal FNR function. The cysteine residue which is centrally located in the three-cysteine cluster (Cys-Ala-Ile-His-Cys-Gln-Asp-Cys) was also shown to be essential for FNR activity. Possible mechanisms by which this cysteine residue might function in the response of FNR to anaerobiosis are discussed. [ABSTRACT FROM AUTHOR]

The FNR protein of E. coli is a transcriptional activator required for the expression of genes involved in anaerobic respiratory pathways. Site-directed mutagenesis was used to alter three amino acids in the recognition helix of the putative DNA-binding domain of FNR, with the aim of changing its specificity to that of the cyclic AMP receptor protein (CRP). In the presence of the mutant protein (FNR-215) expression of the lac operon was activated during anaerobiosis and unaffected by glucose. FNR-215 did not have a uniform effect on the expression of other cAMP-CRP-dependent genes, but the results demonstrate the fundamental similarity between FNR- and CRP-mediated transcriptional activation. [ABSTRACT FROM AUTHOR]

The repressor of the pdhR-aceEF-lpd operon of Escherichia coli, PdhR, was amplified to 23% of total cell protein and purified to homogeneity by heparin-agarose and cation-exchange chromatography. The purified protein is a monomer (Mr, 29 300) which binds specifically to DNA fragments containing the pdh promoter (Ppdh) in the absence of pyruvate. Thepdh operator was identified by DNase I footprinting as a region of hyphenated dyad symmetry, +11AATTGGTaagACCAATT+27, situated just downstream of the transcript start site. In vitro transcription from Ppdh was repressed > 1000-fold by PdhR and this repression was antagonized in a concentration-dependent manner by its co-effector, pyruvate. Studies on RNA polymerase binding at Ppdh showed that RNA polymerase protects the -44 to +21 region in the absence of PdhR, but no RNA polymerase binding or protection upstream of +9 could be detected in the presence of PdhR. It is concluded that PdhR represses transcription by binding to an operator site centred at +19 such that effective binding of RNA polymerase is prevented. [ABSTRACT FROM AUTHOR]

The yfiD gene of Escherichia coli has an unusual promoter architecture in which an FNR dimer located at -93.5 inhibits transcription activation mediated by another FNR dimer bound at the typical class II position (-40.5). In vitro transcription from the yfiD promoter indicated that FNR alone can downregulate yfiD expression. Analysis of yfiD::lac reporters showed that five turns of the DNA helix between FNR sites was optimal for downregulation. FNR heterodimers, in which one subunit carried a defective repression surface, revealed that the upstream subunit of the -40.5 dimer and the downstream subunit of the -93.5 dimer were most important for downregulating yfiD expression. Deletion of the C-terminal domain of the alpha-subunit of RNA polymerase (RNAP) did not affect FNR-mediated repression, suggesting that repression is mediated through FNR-FNR and not FNR-RNAP interactions. Maximum yfiD::lac expression was observed in cultures exposed to 10 microM oxygen. More or less oxygen reduced expression dramatically. This pattern of response was dependent on the combination of a high-affinity site at the activating class II position and a lower affinity site at the upstream position.

The commercially important bacterium Lactococcus lactis contains two FNR-like proteins (FlpA and FlpB) which have a high degree of identity to each other and to the FLP of Lactobacillus casei. FlpA was isolated from a GST-FlpA fusion protein produced in Escherichia coli. Like FLP, isolated FlpA is a homodimeric protein containing both Zn and Cu. However, the properties of FlpA were more like those of the E. coli oxygen-responsive transcription factor FNR than the FLP of L. casei. As prepared FlpA recognized an FNR site (TTGAT-N4-ATCAA) but not an FLP site (CCTGA-N4-TCAGG) in band-shift assays. In contrast to FLP, DNA binding by FlpA did not require the formation of an intramolecular disulphide bond. However, despite containing only two cysteine residues per monomer, FlpA was able to acquire an FNR-like, oxygen-labile [4Fe 4S] cluster. But, whereas the incorporation of a [4Fe 4S] cluster into FNR enhances interaction with target DNA, it abolished DNA binding by FlpA. An FlpA variant (FlpA') with an N-terminal region designed to be more FLP-like failed to incorporate an iron-sulphur cluster but could now form an intramolecular disulphide. This simple example of protein engineering, converting an oxygen-labile [4Fe 4S] containing FNR-like protein into a dithiol-disulphide FLP-like redox sensor demonstrates the versatility of the basic CRP structure. Attempts to demonstrate an FlpA-based aerobic-anaerobic switch in the heterologous host E. coli were unsuccessful. However, studies with a series of FNR-dependent lac reporter fusions in strains of E. coli expressing flpA or flpB revealed that both homologues were able to activate expression of FNR-dependent promoters in vivo but only when positioned 61 base pairs upstream of the transcription start.

Global regulatory circuits of the type mediated by CRP and FNR in Escherichia coli were sought in Lactococcus lactis to provide a basis for redirecting carbon metabolism to specific fermentation products. Using a polymerase chain reaction (PCR) approach, two genes (flpA and flpB) encoding FNR-like proteins (FlpA and FlpB) with the potential for mediating a dithiol-disulphide-dependent regulatory switch, were identified. Transcript analysis indicated that they are distal genes of two paralogous operons, orfX-orfY-flp, in which the orfX and orfY genes were predicted to encode binding domain components of cation ATPases and storage proteins respectively. The corresponding promoters were each associated with a potential FNR site (TTGAT----ATCAA) at positions +4.5 (flpA operon) and -42.5 (flpB operon), suggesting that the respective operons might be negatively and positively autoregulated. The incomplete open reading frames (orfWA/B) located upstream of each operon were predicted to encode additional components of paralogous cation ATPases. No phenotypic effects were detected in flpA and flpB single mutants, but the double mutant had a lower intracellular zinc content, an increased sensitivity to hydrogen peroxide and an altered polypeptide profile (as determined by two-dimensional gel electrophoresis): formate production was not affected. It was concluded tentatively that FlpA and FlpB regulate overlapping modulons, including systems concerned with zinc uptake, in response to metal ion or oxidative stress.

The repressor of the pdhR-aceEF-lpd operon of Escherichia coli, PdhR, was amplified to 23% of total cell protein and purified to homogeneity by heparin-agarose and cation-exchange chromatography. The purified protein is a monomer (M(r) 29,300) which binds specifically to DNA fragments containing the pdh promoter (Ppdh) in the absence of pyruvate. The pdh operator was identified by DNase I footprinting as a region of hyphenated dyad symmetry, +11AATTGGTaagACCAATT+27, situated just downstream of the transcript start site. In vitro transcription from Ppdh was repressed > 1000-fold by PdhR and this repression was antagonized in a concentration-dependent manner by its co-effector, pyruvate. Studies on RNA polymerase binding at Ppdh showed that RNA polymerase protects the -44 to +21 region in the absence of PdhR, but no RNA polymerase binding or protection upstream of +9 could be detected in the presence of PdhR. It is concluded that PdhR represses transcription by binding to an operator site centred at +19 such that effective binding of RNA polymerase is prevented.

Transcript mapping and studies with lacZ translational fusions have shown that the pdhR gene (formerly genA) is the proximal gene of the pdhR-aceE-aceF-lpd operon encoding the pyruvate dehydrogenase (PDH) complex of Escherichia coli. A pdhR-lpd read-through transcript (7.4 kb) initiating at the pyruvate-inducible pdh promoter, and a smaller lpd transcript (1.7 kb) initiating at the independent lpd promoter, were identified. Evidence showing that the pdhR gene product negatively regulates the synthesis of the PdhR protein and the PDH complex via the pdh promoter was obtained, with pyruvate (or a derivative) serving as the putative inducing coeffector. The partially purified PdhR protein was also found to specifically retard and protect DNA fragments containing the pdh promoter region. The pdh promoter was not strongly controlled by ArcA, FNR or CRP.

FNR, the activator of anaerobic respiratory genes of Escherichia coli, has previously only been isolated as a protein of Mr 29,000, which lacks nine N-terminal amino acid residues. The underlying proteolytic events have been studied with the aim of isolating intact FNR and determining whether cleavage is the result of a physiologically significant intracellular processing mechanism or proteolytic degradation during isolation. The FNR protein was present in aerobically and anaerobically grown bacteria as the intact protein (Mr 30,000). Proteolysis only occurred during and shortly after disruption of the bacteria. The production of FNR (Mr 29,000) must therefore be regarded as an isolation artefact. The proteolysis was caused by a protease which is located outside the cytoplasmic membrane or activated upon disruption of the membrane. Protease inhibitors directed against serine, cysteine or metalloproteases failed to prevent cleavage of FNR. In E. coli strain CAG627, proteolysis was greatly reduced making it possible to isolate FNR of Mr 30,000. The N-terminal sequence of FNR (Mr 30,000) was identical to that predicted from the fnr gene starting with the initiating methionine residue and including a four-cysteine cluster (16)Cys-X3-Cys-X2-Cys-X5-Cys(29).