Biomedical subjects

J R Guest

Publications and source records attributed to J R Guest.

At least 55 records · Page 3Linked to original sources

PubMed1996

Lipoyl domain-based mechanism for the integrated feedback control of the pyruvate dehydrogenase complex by enhancement of pyruvate dehydrogenase kinase activity.

To conserve carbohydrate reserves, the reaction of the pyruvate dehydrogenase complex (PDC) must be down-regulated when the citric acid cycle is provided sufficient acetyl-CoA. PDC activity is reduced primarily through increased phosphorylation of its pyruvate dehydrogenase (E1) component due to E1 kinase activity being markedly enhanced by elevated intramitochondrial NADH:NAD+ and acetyl-CoA:CoA ratios. A mechanism is evaluated in which enhanced kinase activity is facilitated by the build-up of the reduced and acetylated forms of the lipoyl moieties of the dihydrolipoyl acetyltransferase (E2) component through using NADH and acetyl-CoA in the reverse of the downstream reactions of the complex. Using a peptide substrate, kinase activity was stimulated by these products, ruling out the possibility kinase activity is increased due to changes in the reaction state of its substrate, E1 (thiamin pyrophosphate). Each E2 subunit contains two lipoyl domains, an NH2-terminal (L1) and the inward lipoyl domain (L2), which were individually produced in fully lipoylated forms by recombinant techniques. Although reduction and acetylation of the L1 domain or free lipoamide increased kinase activity, those modifications of the lipoate of the kinase-binding L2 domain gave much greater enhancements of kinase activity. The large stimulation of the kinase generated by acetyl-CoA only occurred upon addition of the transacetylase-catalyzing (lipoyl domain-free) inner core portion of E2 plus a reduced lipoate source, affirming that acetylation of this prosthetic group is an essential mechanistic step for acetyl-CoA enhancing kinase activity. Similarly, the lesser stimulation of kinase activity by just NADH required a lipoate source, supporting the need for lipoate reduction by E3 catalysis. Complete enzymatic delipoylation of PDC, the E2-kinase subcomplex, or recombinant L2 abolished the stimulatory effects of NADH and acetyl-CoA. Retention of a small portion of PDC lipoates lowered kinase activity but allowed stimulation of this residual kinase activity by these products. Reintroduction of lipoyl moieties, using lipoyl protein ligase, restored the capacity of the E2 core to support high kinase activity along with stimulation of that activity up to 3-fold by NADH and acetyl-CoA. As suggested by those results, the enhancement of kinase activity is very responsive to reductive acetylation with a half-maximal stimulation achieved with approximately 20% of free L2 acetylated and, from an analysis of previous results, with acetylation of only 3-6 of the 60 L2 domains in intact PDC. Based on these findings, we suggest that kinase stimulation results from modification of the lipoate of an L2 domain that becomes specifically engaged in binding the kinase. In conclusion, kinase activity is attenuated through a substantial range in response to modest changes in the proportion of oxidized, reduced, and acetylated lipoyl moieties of the L2 domain of E2 produced by fluctuations in the NADH:NAD+ and acetyl-CoA:CoA ratios as translated by the rapid and reversible E3 and E2 reactions.

Acetyl Coenzyme A↗

PubMed1996

FNR-DNA interactions at natural and semi-synthetic promoters.

Two rapid and convenient methods have been developed for the amplification and purification of FNR, the anaerobic transcription regulator of Escherichia coli. The overproduced proteins resemble wild-type FNR in their basic properties: oligomeric state, iron contents (up to 2.7 atoms per monomer), DNA-binding affinities and ability to activate transcription. However, unlike previous preparations, FNR could be isolated in a form containing up to 0.25 atoms of acid-labile sulphur per monomer. Incorporation of iron increased the Mr of FNR from 28,000 to 40,000. Under anaerobic conditions, reconstituted FNR exhibited absorption maxima at 315 nm and 420 nm, which were replaced by a broad absorbance from 380 to 440 nm under aerobic conditions. These observations indicate that FNR contains one redox-sensitive [3Fe 4S] or [4Fe 4S] centre per monomer. Footprints of FNR-dependent promoters (ansB, fdn, fnr, narG, pflP6, pflP7 and nirB) showed protection at all of the predicted FNR sites except the pflP7 (-57.5), ansB (-74.5) and nirB (-89.5) sites. An unpredicted second binding site was detected at -57.5 in the narG promoter. Hypersensitive sites within regions of FNR protection indicated that FNR bends DNA in a similar way to CRP. Promoters containing binding sites for FNR (FF), CRP (CC) or hybrid sites (CF or FC) were footprinted with FNR and two derivatives (FNR-610 and FNR-573) which activate the CCmelR promoter in vivo. FNR preferentially protected the FNR site (FF) whereas FNR-610 preferred CC and FNR-573 interacted with equal affinity at all sites.

Amino Acid Sequence↗

PubMed1996

The ndh-binding protein (Nbp) regulates the ndh gene of Escherichia coli in response to growth phase and is identical to Fis.

The ndh gene that encodes the non-proton-translocating NADH dehydrogenase II of Escherichia coli is anaerobically repressed by FNR. However, in the absence of FNR, ndh expression is enhanced by anaerobic growth in media containing amino acids. Two potential regulatory proteins that may be associated with this activation have previously been detected, Arr (amino acid response regulator) and Nbp (ndh-binding protein). Studies with the heat-stable Nbp have now shown that it is present in E. coli grown both aerobically and anaerobically in rich and minimal media, indicating that it is not specifically associated with the anaerobic enhancement of ndh expression. The Nbp activity of aerobic cultures was maximal during exponential growth phase (when ndh promoter activity is minimal) but fell rapidly as cultures entered stationary phase and ndh expression increased. Protein purification and mutant studies have further shown that Nbp is identical to the Fis protein (factor for inversion stimulation). Three major and two minor Nbp (Fis)-binding sites have been identified in the ndh promoter by gel retardation and DNase I footprinting. The major sites are centred at -123, -72 and +51, in decreasing order of binding affinity. At low concentrations, Nbp (Fis) increased transcription from the ndh promoter by up to 25%, whereas at higher concentrations it prevented RNA polymerase (RNAP) binding and open complex formation. Consequently, Nbp (Fis) can both activate and repress transcription from the ndh promoter. The results suggest that Nbp (Fis) serves to ensure that the energetically efficient proton-translocating NADH dehydrogenase I is used in preference to the non-proton translocating NADH dehydrogenase II during periods of rapid growth, by repressing expression of the ndh gene.

Base Sequence↗

PubMed1995

Identification of the ferroxidase centre of Escherichia coli bacterioferritin.

The bacterioferritin (BFR) of Escherichia coli takes up iron in the ferrous form and stores it within its central cavity as a hydrated ferric oxide mineral. The mechanism by which oxidation of iron (II) occurs in BFR is largely unknown, but previous studies indicated that there is ferroxidase activity associated with a site capable of forming a dinuclear-iron centre within each subunit [Le Brun, Wilson, Andrews, Harrison, Guest, Thomson and Moore (1993) FEBS Lett. 333, 197-202]. We now report site-directed mutagenesis experiments based on a putative dinuclear-metal-ion-binding site located within the BFR subunit. The data reveal that this dinuclear-iron centre is located at a site within the four-alpha-helical bundle of each subunit of BFR, thus identified as the ferroxidase centre of BFR. The metal-bound form of the centre bears a remarkable similarity to the dinuclear-iron sites of the hydroxylase subunit of methane mono-oxygenase and the R2 subunit of ribonucleotide reductase. Details of how the dinuclear centre of BFR is involved in the oxidation mechanism were investigated by studying the inhibition of iron (II) oxidation by zinc (II) ions. Data indicate that zinc (II) ions bind at the ferroxidase centre of apo-BFR in preference to iron (II), resulting in a dramatic reduction in the rate of oxidation. The mechanism of iron (II) oxidation is discussed in the light of this and previous work.

Amino Acid Sequence↗

PubMed1995

The Leeuwenhoek Lecture, 1995. Adaptation to life without oxygen.

The Earth was populated by anaerobic organisms for at least a thousand million years before the atmosphere became oxygenated and aerobes could evolve. Many bacteria like Escherichia coli retain the ability to grow under both aerobic and anaerobic conditions. Recent studies have revealed some global regulatory mechanisms for activating or repressing the expression of relevant genes in response to oxygen availability. These mechanisms ensure that the appropriate metabolic mode is adopted when bacteria switch between aerobic and anaerobic environments.

Adaptation, Biological↗

PubMed1995

Iron(II) oxidation by H chain ferritin: evidence from site-directed mutagenesis that a transient blue species is formed at the dinuclear iron center.

The iron storage molecule, ferritin, consists of an iron core surrounded by a shell of 24 protein subunits, which, in mammals, are of two types, H and L. Prior to storage of iron as a hydrous ferric oxide within the protein shell, Fe(II) is catalytically oxidized at dinuclear centers within H chains. When 48 Fe(II) atoms/molecule were added to 1 microM recombinant human H chain apoferritin (apo-HuHF), in 0.1 M Mes (pH 6.5), oxidation was 80% complete within about 0.2 s while 99% of the Fe(II) was oxidized within 10 s. A broad visible absorption band (400-800 nm, with a maximum at 650 nm) appeared during the fast phase of Fe(II) oxidation. It reached a plateau at 0.2-0.3 s and then declined while Fe(II) oxidation proceeded to completion and absorbance in the near-UV (300-400 nm) increased. The transient visible species was not observed when Tyr-34 was replaced by phenylalanine or when other conserved amino acids at the ferroxidase centers were substituted by residues which are unable to bind iron or which alter the charge balance. When a second increment of 48 iron atoms was added, 10 min after the first, the visible absorbance was absent and the rate of oxidation slower. Restoration of full oxidative activity took over 24 h. The data indicate that the fast oxidation of Fe(II) by apo-HuHF and the transient visible absorbance associated with it are due to Fe(II) oxidation at the ferroxidase centers.

Binding Sites↗

PubMed1995

Site-directed replacement of the coaxial heme ligands of bacterioferritin generates heme-free variants.

The bacterioferritin (BFR) of Escherichia coli is a heme-containing iron storage molecule. It is composed of 24 identical subunits, which form a roughly spherical protein shell surrounding a central iron storage cavity. Each of the 12 heme moieties of BFR possesses bis-methionine axial ligation, a heme coordination scheme so far only found in bacterioferritins. Members of the BFR family contain three partially conserved methionine residues (excluding the initiating methionine) and in this study each was substituted by leucine and/or histidine. The Met52 variants were devoid of heme, whereas the Met31 and Met86 variants possessed full heme complements and were spectroscopically indistinguishable from wild-type BFR. The heme-free Met52 variants appeared to be correctly assembled and were capable of accumulating iron both in vivo and in vitro. No major differences were observed in the overall rate of iron accumulation for BFR-M52H, BFR-M52L, and the wild-type protein. The iron contents of the Met52 variants, as isolated, were at least 4 times greater than for wild-type BFR. This study is consistent with the reported location of the BFR heme site at the 2-fold axis and shows that heme is unnecessary for BFR assembly and iron uptake.

Amino Acid Sequence↗

PubMed1995

Spectroscopic characterisation of an aconitase (AcnA) of Escherichia coli.

A spectroscopic study of an aconitase, AcnA, from Escherichia coli is presented. The amino acid sequence of AcnA has 53% identity with mammalian cytosolic aconitase (c-aconitase) which is the translational regulator known as iron regulatory factor (IRF). In the [3Fe-4S](+)-containing, inactive state, AcnA displays an EPR signal which is not unlike the corresponding signal from mammalian mitochondrial aconitase (m-aconitase) but is even more similar to the signal from c-aconitase. This is perhaps related to the greater similarity of the AcnA amino acid sequence with c-aconitase. Magnetic circular dichroism (MCD) spectroscopy has revealed that the electronic structure of the [3Fe-4S] cluster of AcnA must be similar to, but not identical to that of m-aconitase. Whilst the [Fe-4S] clusters from both of these enzymes display some features in their MCD spectra common to [3Fe-4S] clusters in general, their spectra overall are unique and indicate that the Fea atom of the [4Fe-4S] form is not the only unusual feature of the [Fe-S] clusters of aconitases. Active [4Fe-4S]-containing AcnA can be reduced to yield an EPR signal due to a [4Fe-4S]+ cluster which is indistinguishable from the signals from the [4Fe-4S]+ cluster in the mammalian enzymes. However, in contrast to the mammalian enzymes, the EPR signals of the cluster in AcnA are not significantly perturbed upon the addition of substrate. Furthermore, the catalytic activity of [Fe-4S](2+)-containing AcnA is fivefold higher than that of m-aconitase. The mechanistic implications of these data are discussed. A novel S = 1/2 EPR signal with g approximately 2 was observed in AcnA upon treatment with EDTA. The species giving rise to this signal is proposed to be an intermediate in cluster deconstruction.

Aconitate Hydratase↗

PubMed1995

Purification and properties of the lipoate protein ligase of Escherichia coli.

Lipoate is an essential component of the 2-oxoacid dehydrogenase complexes and the glycine-cleavage system of Escherichia coli. It is attached to specific lysine residues in the lipoyl domains of the E2p (lipoate acetyltransferase) subunit of the pyruvate dehydrogenase complex by a Mg(2+)- and ATP-dependent lipoate protein ligase (LPL). LPL was purified from wild-type E. coli, where its abundance is extremely low (< 10 molecules per cell) and from a genetically amplified source. The purified enzyme is a monomeric protein (M(r) 38,000) which forms irregular clusters of needle-like crystals. It is stable at -20 degrees C, but slowly oxidizes to an inactive form containing at least one intramolecular disulphide bond at 4 degrees C. The inactive form could be re-activated by reducing agents or by an as-yet unidentified component (reactivation factor) which is resolved from LPL at the final stage of purification. The pI is 5.80, and the Km values for ATP, Mg2+ and DL-lipoate were determined. Selenolipoate and 6-thio-octanoate were alternative but poorer substrates. Lipoylation was reversibly inhibited by the 6- and 8-seleno-octanoates and 8-thio-octanoate, which reacted with the six cysteine thiol groups of LPL. LPL was inactivated by Cu2+ ions in a process that involved the formation of inter- and intra-molecular disulphide bonds. Studies with lplA mutants lacking LPL activity indicated that E. coli possesses another distinct lipoylation system, although no such activity could be detected in vitro.

Amino Acid Sequence↗

PubMed1995

Three-dimensional structure of a lipoyl domain from the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The structure of a lipoyl domain from the pyruvate dehydrogenase multienzyme complex of Escherichia coli has been determined by means of nuclear magnetic resonance spectroscopy. A total of 549 nuclear Overhauser effect distance restraints, 52 phi torsion angle restraints and 16 slowly exchanging amide protons were employed as input for the structure calculations. These were performed using a combined distance geometry-simulated annealing strategy. The domain is a hybrid between the N and C-terminal halves of the first and third lipoyl domains, respectively, of the dihydrolipoyl acetyltransferase component of the E. coli multienzyme complex, representing residues 1 to 33 and 238 to 289 (wild-type numbering). The lipoyl-lysine residue was also replaced by glutamine. Nonetheless, its structure, two four-stranded beta-sheets forming a flattened beta-barrel, closely resembles that of the lipoyl domain from the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus determined previously. As before, the lipoylation site is physically exposed in a tight turn in one of the beta-sheets, and the N and C-terminal residues are close together at the other end of the molecule in adjacent strands of the other beta-sheet. Another prominently conserved feature of the structure is the 2-fold axis of quasi-symmetry relating the N and C-terminal halves of the domain. Consistent with the high level of sequence similarity between lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes from many different sources, these results confirm that all lipoyl domains are likely to have closely related structures.

Acetyltransferases↗

PubMed1995

Purification, characterization and mode of action of PdhR, the transcriptional repressor of the pdhR-aceEF-lpd operon of Escherichia coli.

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.

Acetyltransferases↗

PubMed1994

Identification of a fatty acyl responsive regulator (FarR) in Escherichia coli.

FarR (formerly P30) has been identified as a fatty acid and fatty acyl-CoA responsive DNA-binding protein. It is encoded by the farR gene (g30) in the citric acid cycle gene cluster of E. coli (gltA-sdhCDAB-sucABCD-farR). The amplified FarR protein specifically bound to the farR promoter (PfarR) and exhibited weak binding to the citrate synthase and lipoamide dehydrogenase promoters. Binding at PfarR was abolished by long-chain fatty acids and their CoA thioesters. In DNaseI footprints, FarR binding at PfarR protected two sites, each characterised by two related 10-bp direct repeats. It is suggested that FarR autoregulates farR expression and may modulate citric acid cycle expression in response to long-chain fatty acids.

Acyl Coenzyme A↗

PubMed1994

Iron incorporation into ferritins: evidence for the transfer of monomeric Fe(III) between ferritin molecules and for the formation of an unusual mineral in the ferritin of Escherichia coli.

Iron that has been oxidized by H-chain ferritin can be transferred into other ferritin molecules before it is incorporated into mature ferrihydrite iron cores. Iron(III) dimers are formed at the ferroxidase centres of ferritin H chains at an early stage of Fe(II) oxidation. Mössbauer spectroscopic data now show that the iron is transferred as monomeric species arising from dimer dissociation and that it binds to the iron core of the acceptor ferritin. Human H-chain ferritin variants containing altered threefold channels can act as acceptors, as can the ferritin of Escherichia coli (Ec-FTN). A human H-chain ferritin variant with a substituted tyrosine (rHuHF-Y34F) can act as a donor of Fe(III). Since an Fe(III)-tyrosinate (first identified in bullfrog H-chain ferritin) is absent from variant rHuHF-Y34F, the Fe(III) transferred is not derived from this tyrosinate complex. Mössbauer parameters of the small iron cores formed within Ec-FTN are significantly different from those of mammalian ferritins. Analysis of the spectra suggests that they are derived from both ferrihydrite and non-ferrihydrite components. This provides further evidence that the ferritin protein shell can influence the structure of its iron core.

Escherichia coli↗

PubMed1994

Direct observation of the iron binding sites in a ferritin.

X-Ray analysis of the ferritin of Escherichia coli (Ec-FTN) and of Ec-FTN crystals soaked in (NH4)2Fe(SO4)2 has revealed the presence of three iron-binding sites per subunit. Two of these form a di-iron site in the centre of the subunit as has been proposed for the 'ferroxidase centres' of human ferritin H chains. This di-iron site, lying within the 4-alpha-helix bundle, resemble those of ribonucleotide reductase, methane monoxygenase and haemerythrin. The third iron is bound by ligands unique to Ec-FTN on the inner surface of the protein shell. It is speculated that this state may represent the nucleation centre of a novel type of Fe(III) cluster, recently observed in Ec-FTN.

Bacterial Proteins↗

PubMed1994

Modulation of the oxidation-reduction potential of the flavin in lipoamide dehydrogenase from Escherichia coli by alteration of a nearby charged residue, K53R.

The epsilon-amino group of a lysine residue occupies a position within bonding distance of the flavin N5 and the bound NADPH pyridinium C4' in glutathione reductase, and it has been suggested that this positive charge influences the redox potential of the FAD [Pai & Schulz (1983) J. Biol. Chem. 258, 1752]. A conserved lysine residue occupies a similar position in lipoamide dehydrogenase. This residue has been replaced by an arginine in lipoamide dehydrogenase from Escherichia coli to give K53R. The spectral and redox properties of the FAD in K53R as well as the interaction of the flavin with bound NAD+ are profoundly affected by the change. K53R does not catalyze either the dihydrolipoamide-NAD+ or the NADH-lipoamide reactions except at very low concentrations of the reducing substrate. The absorbance spectrum of K53R in the visible and near-ultraviolet is little changed from that of wild-type enzyme, but in contrast, the spectrum of K53R is sensitive to pH with an apparent pKa = 7.0. Unlike the wild-type enzyme, the binding of beta-NAD+ to K53R alters the spectrum and indicates an apparent Kd = 7.0 microM at pH 7.6. The flavin fluorescence is partially quenched, and the visible and near-ultraviolet circular dichroism spectrum is changed by beta-NAD+. K53R is extensively reduced (mostly EH4) by 2 equiv of dihydrolipoamide/FAD while the wild-type enzyme is only partially reduced (mostly EH2). The rate of this reduction is lowered by approximately 3-fold relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗

PubMed1994

Two genetically-distinct and differentially-regulated aconitases (AcnA and AcnB) in Escherichia coli.

An acnA mutant of Escherichia coli was constructed by replacing the chromosomal acnA gene by an internally deleted derivative containing a kanR cassette. Southern and Western blotting confirmed that the acnA gene had been replaced by the disrupted gene and that the aconitase A protein was no longer expressed. However, the mutant failed to exhibit the anticipated glutamate auxotrophy and it retained a residual aconitase activity. This activity was due to an analogous unstable enzyme(s) designated aconitase B. Studies on the regulation of aconitase A synthesis using an acnA-lacZ translational fusion showed that the acnA gene resembles other citric acid cycle genes in being subject to CRP-mediated catabolite repression and ArcA-mediated anaerobic repression. In addition to being activated by the SoxRS oxidative stress regulatory system, the acnA gene appeared to be activated by the ferric uptake regulator (Fur). It was concluded that the acnA gene belongs to at least four global regulatory networks, crp, arcA, fur and soxRS. In contrast, the aconitase B activity decreased after exposure to oxidative stress and was less affected by anaerobiosis. Comparable studies with the fumarase genes (fumA, B and C) indicated that fumA (encoding the unstable aerobic iron-sulphur-containing fumarase) is activated by the ferric uptake regulator (Fur) and fumC (encoding the stable fumarase) is activated by the SoxRS oxidative stress regulatory system.

Aconitate Hydratase↗

PubMed1994

The pdhR-aceEF-lpd operon of Escherichia coli expresses the pyruvate dehydrogenase complex.

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.

Acetyltransferases↗

PubMed1994

Regulation of transcription at the ndh promoter of Escherichia coli by FNR and novel factors.

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 deletion strain was enhanced by anaerobic growth in rich medium or minimal medium supplemented with amino acids. Furthermore, two proteins (M(r) 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.

Amino Acids↗