Fnr Gene Research Articles

Microaerobic conditions enhance laccase production from Rheinheimera sp. in an economical medium.

Statistical optimization of aeration conditions viz. aerobic, microaerobic and anaerobic, was performed using response surface methodology (RSM) utilizing soybean meal as medium to enhance the production of laccase from Rheinheimera sp. Maximum laccase yield (18.48 × 105U/L) was obtained under microaerobic (static) conditions sustained for 12h in tandem with 26h aerobically (150rpm) grown culture, which was 17.03-fold higher than laccase production in the starting M162 medium under aerobic conditions (150rpm). The reduction in incubation time from 72 to 38h and utilization of cost-effective soybean meal as medium, which is easily available from local market, have provided a promising, eco-friendly method of laccase enzyme production. Enhanced expression of laccase gene under microaerobic conditions corresponded to the increased expression of fnr (fumarate nitrate reductase) gene, the oxygen sensing global regulator. The putative FNR-binding site upstream of laccase transcription initiation site was predicted to play an imperative role in Rheinheimera sp. adaptation from aerobic to microaerobic conditions and for enhanced laccase production.

Recoding anaerobic regulator fnr of Salmonella Typhimurium attenuates it's pathogenicity

AimsHow recoding of fnr, an anaerobic regulatory gene, affects pathogenicity related parameters of Salmonella Typhimurium (STM). Methods and resultsThe fnr gene was recoded by substituting all of it's codons with synonymous rare codons of STM. Recoding fnr gene severely reduced the ability of the recoded mutant to compete with wild strain under nutrient depletion condition. Mutants were also less motile than the wild strain and their biofilm forming ability was significantly decreased as compared to wild strain. The recoded strain showed significant reduced survival within murine macrophages (RAW264.7) and monocyte derived macrophage of poultry origin. The colonisation ability of recoded mutant in liver and spleen of mice on day 5 of post infection was significantly reduced. The recoded strain exhibited significant reduction in faecal shedding on day 1 and 5 after infection. ConclusionsOur study showed that recoding the anaerobic regulator fnr of STM significantly compromised its growth, decreased motility, biofilm forming ability and survival within macrophages. Further, the recoded fnr strain showed reduced colonisation ability and faecal shedding in mice. Thus, these findings highlight that recoding the global anaerobic regulator fnr of Salmonella Typhimurium attenuates its pathogenicity.

Crp/fnr family protein binds to promoters of atxA and sodmn genes that regulate the expression of exotoxins in Bacillus anthracis

Bacillus anthracis produces a tripartite exotoxin, which is regulated by AtxA. Sodmn is constitutively expressed during invasion. Crp/Fnr family transcriptional regulators are known to bind promoters of toxin regulators as well as constitutively expressed genes during pathogenesis. B. anthracis fnr gene was cloned, over-expressed in E. coli and recombinant protein was purified. Oligomeric nature of recombinant rFnr protein was studied by diamide treatment and DTT reduction. DNA binding of rFnr protein was studied by EMSA. We observed that rFnr exists in both monomeric and oligomeric forms. It was found that rFnr was able to oligomerize after diamide treatment which was reversible through DTT reduction. Promoter regions of atxA and sodmn show binding to monomeric form of rFnr, however, dimeric form was unable to bind. Fnr might be playing a role in regulation of toxin gene expression via regulation of atxA gene. It can also be involved in regulation of pathogenesis by regulating the sodmn expression. Oligomerization can act as an ON/OFF switch for the Fnr mediated regulation.

Regulatory Role of fnr Gene in Growth and tolA Gene Expression in Salmonella Typhimurium

Background: Salmonella Typhimurium (S.Typhimurium) adapts to the broad fluctuations of oxygen concentrations encountered in the host. The transition from aerobic to microaerobic/anaerobic condition encountered in the intestine is mainly regulated by fumarate and nitrate reductase (fnr) regulatory gene and aerobic respiratory control A (arcA) gene. Aim is to appraise the role of fnr gene under anaerobic conditions.Methods: In this study, we deleted fnr gene from S.Typhimurium using lambda red-recombinase mediated gene knockout protocol. Further carried out in vitro characterization and analyzed the differential protein expression in wild type (WT) and isogenic Δfnr null mutant (Δfnr) using SDS-PAGE and MALDI-TOF mass spectrometry under anaerobic conditions.Result: In growth competition, WT strain outcompeted the Δfnr and biofilm-forming ability of Δfnr was significantly reduced compared to WT strain. Swimming motility was reduced in Δfnr strain. Besides, differential protein expression revealed the global changes in the expression of many proteins in fnr strain. One differentially expressed protein was identified as TolA, an inner membrane envelope protein. It points out that fnr may regulate the genes responsible for motility and biofilm formation. FNR protein positively regulates TolA, which is important for bacterial virulence, maintenance of membrane integrity, LPS production and replication of bacteria.

Heterologous expression of the gene for chlorite dismutase from Ideonella dechloratans is induced by an FNR-type transcription factor.

Regulation of the expression of the gene for chlorite dismutase (cld), located on the chlorate reduction composite transposon of the chlorate reducer Ideonella dechloratans, was studied. A 200 bp upstream sequence of the cld gene, and mutated and truncated versions thereof, was used in a reporter system in Escherichia coli. It was found that a sequence within this upstream region, which is nearly identical to the canonical FNR‐binding sequence of E. coli, is necessary for anaerobic induction of the reporter gene. Anaerobic induction was regained in an FNR‐deficient strain of E. coli when supplemented either with the fnr gene from E. coli or with a candidate fnr gene cloned from I. dechloratans. In vivo transcription of the suggested fnr gene of I. dechloratans was demonstrated by qRT‐PCR. Based on these results, the cld promoter of I. dechloratans is suggested to be a class II‐activated promoter regulated by an FNR‐type protein of I. dechloratans. No fnr‐type genes have been found on the chlorate reduction composite transposon of I. dechloratans, making anaerobic upregulation of the cld gene after a gene transfer event dependent on the presence of an fnr‐type gene in the recipient.

Importance of fumarate and nitrate reduction regulatory protein for intestinal proliferation of Vibrio vulnificus.

The sepsis caused by Vibrio vulnificus is characterized by an average incubation period of 26 h and a high mortality rate exceeding 50%. The fast growth and dissemination of V. vulnificus in vivo lead to poor clinical outcomes in patients. Therefore, elucidation of the proliferation mechanisms of this organism in vivo may lead to the development of an effective therapeutic strategy. In this study, we focused on the low oxygen concentration in the intestinal milieu because of its drastic difference from that in air. Fumarate and nitrate reduction regulatory protein (FNR) is known to be a global transcriptional regulator for adaptation to anaerobic conditions in various bacteria. We generated a strain of V. vulnificus in which the fnr gene was replaced with an erythromycin resistance gene (fnr::erm mutant). When the fnr::erm mutant was tested in a growth competition assay against the wild-type (WT) in vivo, the competitive index of fnr::erm mutant to WT in the intestinal loop and liver was 0.378 ± 0.192 (mean ± SD) and 0.243 ± 0.123, respectively. These data suggested that FNR is important for the proliferation of V. vulnificus in the intestine to achieve a critical mass to be able to invade the systemic circulation.

Metabolic regulation of an fnr gene knockout Escherichia coli under oxygen limitation

The effect of fnr gene knockout on the metabolism of Escherchia coli was investigated under microaerobic condition based on gene expressions, enzyme activities, and intracellular metabolic fluxes. Under micro-aerobic condition, the flux through Pfl and Frd were reduced for the mutant, and the decreased flux through Pfl may have caused accumulation of PYR, which increased the flux through LDH. The fnr gene knockout caused arcA to be down-regulated, and thus the TCA cycle was activated, and cyoA and cydB genes were up-regulated. The down-regulation of arcA caused lpdA to be up-regulated where the flux through PDHc increased. The fnr gene knockout indirectly affected cra gene transcript level to be decreased, which in turn caused the glycolysis genes to be up-regulated, which corresponded to the increase in the specific glucose consumption rate. The fnr gene knockout also caused crp transcript level to be increased, where there might be some relationship between the two due to similar structure and gene sequence.

Metabolic deficiences revealed in the biotechnologically important model bacterium Escherichia coli BL21(DE3).

The Escherichia coli B strain BL21(DE3) has had a profound impact on biotechnology through its use in the production of recombinant proteins. Little is understood, however, regarding the physiology of this important E. coli strain. We show here that BL21(DE3) totally lacks activity of the four [NiFe]-hydrogenases, the three molybdenum- and selenium-containing formate dehydrogenases and molybdenum-dependent nitrate reductase. Nevertheless, all of the structural genes necessary for the synthesis of the respective anaerobic metalloenzymes are present in the genome. However, the genes encoding the high-affinity molybdate transport system and the molybdenum-responsive transcriptional regulator ModE are absent from the genome. Moreover, BL21(DE3) has a nonsense mutation in the gene encoding the global oxygen-responsive transcriptional regulator FNR. The activities of the two hydrogen-oxidizing hydrogenases, therefore, could be restored to BL21(DE3) by supplementing the growth medium with high concentrations of Ni2+ (Ni2+-transport is FNR-dependent) or by introducing a wild-type copy of the fnr gene. Only combined addition of plasmid-encoded fnr and high concentrations of MoO4 2− ions could restore hydrogen production to BL21(DE3); however, to only 25–30% of a K-12 wildtype. We could show that limited hydrogen production from the enzyme complex responsible for formate-dependent hydrogen evolution was due solely to reduced activity of the formate dehydrogenase (FDH-H), not the hydrogenase component. The activity of the FNR-dependent formate dehydrogenase, FDH-N, could not be restored, even when the fnr gene and MoO4 2− were supplied; however, nitrate reductase activity could be recovered by combined addition of MoO4 2− and the fnr gene. This suggested that a further component specific for biosynthesis or activity of formate dehydrogenases H and N was missing. Re-introduction of the gene encoding ModE could only partially restore the activities of both enzymes. Taken together these results demonstrate that BL21(DE3) has major defects in anaerobic metabolism, metal ion transport and metalloprotein biosynthesis.

Aspartate 141 Is the Fourth Ligand of the Oxygen-sensing [4Fe-4S]2+ Cluster of Bacillus subtilis Transcriptional Regulator Fnr

The Bacillus subtilis redox regulator Fnr controls genes of the anaerobic metabolism in response to low oxygen tension. An unusual structure for the oxygen-sensing [4Fe-4S](2+) cluster was detected by a combination of genetic experiments with UV-visible and Mössbauer spectroscopy. Asp-141 was identified as the fourth iron-sulfur cluster ligand besides three Cys residues. Exchange of Asp-141 with Ala abolished functional in vivo complementation of an fnr knock-out strain by the mutagenized fnr gene and in vitro DNA binding of the recombinant regulator FnrD141A. In contrast, substitution of Asp-141 with Cys preserved [4Fe-4S](2+) structure and regulator function.

Regulation of tartrate metabolism by TtdR and relation to the DcuS–DcuR-regulated C4-dicarboxylate metabolism of Escherichia coli

Escherichia coli catabolizes L-tartrate under anaerobic conditions to oxaloacetate by the use of L-tartrate/succinate antiporter TtdT and L-tartrate dehydratase TtdAB. Subsequently, L-malate is channelled into fumarate respiration and degraded to succinate by the use of fumarase FumB and fumarate reductase FrdABCD. The genes encoding the latter pathway (dcuB, fumB and frdABCD) are transcriptionally activated by the DcuS-DcuR two-component system. Expression of the L-tartrate-specific ttdABT operon encoding TtdAB and TtdT was stimulated by the LysR-type gene regulator TtdR in the presence of L- and meso-tartrate, and repressed by O(2) and nitrate. Anaerobic expression required a functional fnr gene, and nitrate repression depended on NarL and NarP. Expression of ttdR, encoding TtdR, was repressed by O(2), nitrate and glucose, and positively regulated by TtdR and DcuS. Purified TtdR specifically bound to the ttdR-ttdA promoter region. TtdR was also required for full expression of the DcuS-DcuR-dependent dcuB gene in the presence of tartrate. Overall, expression of the ttdABT genes is subject to L-/meso-tartrate-dependent induction, and to aerobic and nitrate repression. The control is exerted directly at ttdA and in addition indirectly by regulating TtdR levels. TtdR recognizes a subgroup (L- and meso-tartrate) of the stimuli perceived by the sensor DcuS, which responds to all C(4)-dicarboxylates; both systems apparently communicate by mutual regulation of the regulatory genes.

Detection of Sulfide Release from the Oxygen-sensing [4Fe-4S] Cluster of FNR

The Escherichia coli FNR protein regulates the transcription of >100 genes in response to environmental O2, thereby coordinating the response to anoxia. Under O2-limiting conditions, FNR binds a [4Fe-4S]2+ cluster through four cysteine residues (Cys20, Cys23, Cys29, Cys122). The acquisition of the [4Fe-4S]2+ cluster converts FNR into the transcriptionally active dimeric form. Upon exposure to O2, the cluster converts to a [2Fe-2S]2+ form, generating FNR monomers that no longer bind DNA with high affinity. The mechanism of the cluster conversion reaction and the nature of the released iron and sulfur are of considerable current interest. Here, we report the application of a novel in vitro method, involving 5,5'-dithiobis-(2-nitrobenzoic acid), for determining the oxidation state of the sulfur atoms released during FNR cluster conversion following the addition of O2. Conversion of [4Fe-4S]2+ to [2Fe-2S]2+ clusters by O2 for both native and reconstituted FNR results in the release of approximately 2 sulfide ions per [4Fe-4S]2+ cluster. This demonstrates that the reaction between O2 and the [4Fe-4S]2+ cluster does not require sulfide oxidation and hence must entail iron oxidation.

Aerobic activation of transcription of the anaerobically inducibleEscherichia coli focA-pfloperon by fumarate nitrate regulator

Expression of the anaerobically inducible focA-pfl operon in Escherichia coli was activated nearly sevenfold relative to wild-type under aerobic growth conditions by increasing the dosage of the fnr gene on a pBR322-based plasmid (pCH21). No effect on anaerobic expression levels was observed, suggesting that operon expression under these conditions is maximal. Examination of the complex transcript pattern of the focA-pfl operon confirmed that in strains bearing pCH21 all transcripts, with the exception of the promoter 7 transcript, were up-regulated aerobically. Western analysis of strains bearing pCH21 revealed that the fumarate nitrate regulator (FNR) level was increased approximately ninefold relative to the level in strains bearing a single copy of the fnr gene aerobically, but was only overproduced threefold anaerobically. Analysis of an fnr-lacZ fusion indicated that fnr expression was more strongly negatively autoregulated in anaerobic cells compared with aerobic cells when pCH21 was present. Taken together, these findings suggest that high-level overproduction of FNR is prevented anaerobically by active FNR repressing expression of the fnr gene. Furthermore, transcription from promoter 7 of the focA-pfl operon, which depends on both ArcA-P and FNR, cannot be activated aerobically by overproduction of FNR alone, while promoter 6, which is less dependent on ArcA-P, can be activated under these conditions.

Nitrosomonas europaea expresses a nitric oxide reductase during nitrification.

In this paper, we report the identification of a norCBQD gene cluster that encodes a functional nitric oxide reductase (Nor) in Nitrosomonas europaea. Disruption of the norB gene resulted in a strongly diminished nitric oxide (NO) consumption by cells and membrane protein fractions, which was restored by the introduction of an intact norCBQD gene cluster in trans. NorB-deficient cells produced amounts of nitrous oxide (N2O) equal to that of wild-type cells. NorCB-dependent activity was present during aerobic growth and was not affected by the inactivation of the putative fnr gene. The findings demonstrate the presence of an alternative site of N2O production in N. europaea.

Cloning, sequencing and characterization of Fnr from Klebsiella pneumoniae.

The transcription factor Fnr (fumarate nitrate reductase regulator) globally regulates gene expression in response to oxygen deprivation in Escherichia coli. We report here the cloning and sequencing of the fnr gene from the facultative anaerobic bacterium Klebsiella pneumoniae M5al, another member of the enteric bacteria. The deduced amino acid sequence of K. pneumoniae fnr showed very high similarity (98% amino acid identity) to the Fnr protein from E. coli and contained the four essential cysteine residues which are presumed to build the oxygen-sensing [4Fe4S]+2 center. Transfer of the K. pneumoniae gene to a fnr mutant of E. coli complemented the mutation and permitted synthesis of nitrate reductase and fumarate reductase during anaerobic growth. A gene fusion between K. pneumoniae fnr and glutathione S-transferase was constructed and expressed in E. coli under anaerobic conditions in order to make the protein available in preparative amounts. The overproduced protein was purified by glutathione-Sepharose 4B affinity chromatography in the absence of oxygen, and biochemically characterized.

Overexpression of the FNR protein of Escherichia coli with T7 expression system.

We have used the T7 expression system for expression of E. coli FNR protein. The fnr gene was cloned from its initiation codon ATG into the NdeI site of an expression vector and filamentous phage mGP1-2 was used as a donor of T7 RNA polymerase gene. The level of FNR expression attained by this expression arrangement was about 45% of total cell proteins.

Altering the anaerobic transcription factor FNR confers a hemolytic phenotype on Escherichia coli K12.

The recent outbreaks of Escherichia coli O157-associated food poisoning have focused attention on the virulence determinants of E. coli. Here, it is reported that single base substitutions in the fnr gene encoding the oxygen-responsive transcription regulator FNR (fumarate and nitrate reduction regulator) are sufficient to confer a hemolytic phenotype on E. coli K12, the widely used laboratory strain. The mechanism involves enhancing the expression of a normally dormant hemolysin gene (hlyE) located in the E. coli chromosome. The mutations direct single amino acid substitutions in the activating regions (AR1 and AR3) of FNR that contact RNA polymerase. It is concluded that altering a resident transcription regulator, or acquisition of a competent heterologous regulator, could generate a pool of hemolytic, and therefore more virulent, strains of E. coli in nature.

Transcription activation at class I FNR-dependent promoters: identification of the activating surface of FNR and the corresponding contact site in the C-terminal domain of the RNA polymerase alpha subunit.

A library of random mutations in the Escherichia coli fnr gene has been screened to identify positive control mutants of FNR that are defective in transcription activation at Class I promoters. Single amino acid substitutions at D43, R72, S73, T118, M120, F181, F186, S187 and F191 identify a surface of FNR that is essential for activation which, presumably, makes contact with the C-terminal domain of the RNA polymerase alpha subunit. This surface is larger than the corresponding activating surface of the related transcription activator, CRP. To identify the contact surface in the C-terminal domain of the RNA polymerase alpha subunit, a library of mutations in the rpoA gene was screened for alpha mutants that interfered with transcription activation at Class I FNR-dependent promoters. Activation was reduced by deletions of the alpha C-terminal domain, by substitutions known to affect DNA binding by alpha, by substitutions at E261 and by substitutions at L300, E302, D305, A308, G315 and R317 that appear to identify contact surfaces of alpha that are likely to make contact with FNR at Class I promoters. Again, this surface differs from the surface used by CRP at Class I CRP-dependent promoters.

Role of multiple ArcA recognition sites in anaerobic regulation of succinate dehydrogenase (sdhCDAB) gene expression in Escherichia coli.

Succinate dehydrogenase (sdhCDAB) gene expression in Escherichia coli is negatively regulated by the arcA and fnr gene products during anaerobic cell growth conditions. The controlled synthesis of this sole membrane-bound enzyme of the tricarboxylic acid cycle allows optimal participation in the aerobic electron transport pathway for the generation of energy via oxidative phosphorylation reactions. To understand how ArcA participates in the anaerobic repression of sdhCDAB expression, a family of sdhC-lacZ fusions was constructed and analysed in vivo. DNase I footprint and gel shift assays using purified ArcA protein revealed the location of four distinct and independent ArcA binding sites in the sdhC promoter region. ArcA sites, designated sites 1 and 2, are centred at -205 bp and -119 bp upstream of the sdhC promoter, respectively, whereas ArcA site 3 overlaps the -35 and -10 regions of the sdhC promoter. A fourth ArcA site is centred at +257 bp downstream of the sdhC promoter. They are bound with differing affinity by ArcA and ArcA phosphate. The in vivo studies, in combination with the in vitro studies, indicate that ArcA site 3 is necessary and sufficient for the ArcA-dependent repression of sdhC gene expression, while the DNA region containing ArcA site 2 contributes to maximal gene expression. The DNA-containing ArcA sites 1 and 4 provide minor roles in the ArcA regulation of sdhC expression. Lastly, the Fnr-dependent control of sdhCDAB gene expression was shown to occur independently of the ArcA and to require DNA sequences near the start of sdhC transcription.

Aerobic regulation of the sucABCD genes of Escherichia coli, which encode alpha-ketoglutarate dehydrogenase and succinyl coenzyme A synthetase: roles of ArcA, Fnr, and the upstream sdhCDAB promoter.

The sucABCD genes of Escherichia coli encode subunits for two enzymes of the tricarboxylic acid (TCA) cycle, alpha-ketoglutarate dehydrogenase (sucAB) and succinyl coenzyme A synthetase (sucCD). To examine how these genes are expressed in response to changes in oxygen and carbon availability, a set of sucA-lacZ, sucC-lacZ, sdhCDAB-sucA-lacZ, and sdhC-lacZ fusions were constructed and analyzed in vivo. While the expression of a sucA-lacZ fusion was low under all cell growth conditions tested, the expression of the sucA gene from the upstream sdhC promoter was considerably higher and varied by up to 14-fold depending on the carbon substrate used. Expression of the sdhCDAB-sucA-lacZ fusion varied by fourfold in response to oxygen. In contrast, no expression was seen from a sucC-lacZ reporter fusion, indicating that no promoter immediately precedes the sucCD genes. Taken together, these findings demonstrate that the oxygen and carbon control of sucABCD gene expression occurs by transcriptional regulation of the upstream sdhC promoter. The weaker sucA promoter provides an additional low constitutive level of sucABCD gene expression to supplement transcription from the sdhC promoter. The negative control of sucABCD gene expression seen under anaerobic conditions, like that for the sdhCDAB genes, is provided by the arcA and fnr gene products. These findings establish that the differential expression of eight genes for three of the TCA cycle enzymes in E. coli is controlled from one regulatory element.

Aerobic regulation of isocitrate dehydrogenase gene (icd) expression in Escherichia coli by the arcA and fnr gene products.

Isocitrate dehydrogenase, the icd gene product, has been studied extensively regarding the regulation of enzymatic activity and its relationship to the metabolic flux between the tricarboxylic acid cycle and the glyoxylate bypass. In this study, the transcriptional regulation of icd gene expression was monitored by using an icd-lacZ gene fusion and shown to vary over a 15-fold range in response to changes in oxygen and carbon availability. Anaerobic cell growth resulted in fivefold-lower icd-lacZ expression than during aerobic growth. This negative control is mediated by the arcA and fnr gene products. When different carbon compounds were used for cell growth, icd-lacZ expression varied threefold. The results of continuous cell culture studies indicated that this control may be due to variations in cell growth rate rather than to catabolite repression. DNase I footprinting at the icd promoter revealed a 42-bp ArcA-phosphate-protected region that overlaps the start site of icd transcription. Phosphorylation of ArcA considerably enhanced its binding to DNA, while ArcA-phosphate exhibited an apparent dissociation value of approximately 0.1 microM. Based on these studies, ArcA appears to function as a classical repressor of transcription by binding at a site overlapping the icd promoter during anaerobic cell growth conditions.