Fumarate Nitrate Reduction Regulator Research Articles

FNR-Dependent RmpA and RmpA2 Regulation of Capsule Polysaccharide Biosynthesis in Klebsiella pneumoniae.

Fumarate nitrate reduction regulator (FNR) is a direct oxygen-responsive transcriptional regulator containing an iron-sulfur (Fe–S) cluster. During anaerobic growth, the [4Fe–4S] cluster in FNR (holo-FNR) binds specifically to DNA, whereas exposure to oxygen results in the loss of its DNA-binding activity via oxidation of the [4Fe–4S] cluster. In this study, we aimed to investigate the role of FNR in regulation of capsular polysaccharide (CPS) biosynthesis, serum resistance, and anti-phagocytosis of K. pneumoniae. We found that the CPS amount in K. pneumoniae increased in anaerobic conditions, compared to that in aerobic conditions. An fnr deletion mutant and a site-directed mutant (fnr3CA), with the three cysteines (C20, C23, and C29) replaced with alanines to mimic an FNR lacking the [4Fe-4S] cluster, showed marked increase in CPS amount under anaerobic conditions. A promoter-reporter assay and qRT-PCR confirmed that the transcription of the cps genes was repressed by holo-FNR. In addition, we found that holo-FNR could repress the transcription of rmpA and rmpA2, encoding cps transcriptional activators. Deletion of rmpA or rmpA2 in the Δfnr strain reduced CPS biosynthesis, suggesting that RmpA and RmpA2 participated in the holo-FNR–mediated repression of cps transcription, thereby regulating the CPS amount, serum resistance, and anti-phagocytosis. Taken together, our results provided evidence that RmpA and RmpA2 participated in the holo-FNR–mediated repression of CPS biosynthesis, and resistance to the host defense in response to oxygen availability.

Three Pseudomonas putida FNR Family Proteins with Different Sensitivities to O2

The Escherichia coli fumarate-nitrate reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233, and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR, and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together, the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2.

A bioinformatics insight to rhizobial globins: gene identification and mapping, polypeptide sequence and phenetic analysis, and protein modeling.

Globins (Glbs) are proteins widely distributed in organisms. Three evolutionary families have been identified in Glbs: the M, S and T Glb families. The M Glbs include flavohemoglobins (fHbs) and single-domain Glbs (SDgbs); the S Glbs include globin-coupled sensors (GCSs), protoglobins and sensor single domain globins, and the T Glbs include truncated Glbs (tHbs). Structurally, the M and S Glbs exhibit 3/3-folding whereas the T Glbs exhibit 2/2-folding. Glbs are widespread in bacteria, including several rhizobial genomes. However, only few rhizobial Glbs have been characterized. Hence, we characterized Glbs from 62 rhizobial genomes using bioinformatics methods such as data mining in databases, sequence alignment, phenogram construction and protein modeling. Also, we analyzed soluble extracts from Bradyrhizobium japonicum USDA38 and USDA58 by (reduced + carbon monoxide (CO) minus reduced) differential spectroscopy. Database searching showed that only fhb, sdgb, gcs and thb genes exist in the rhizobia analyzed in this work. Promoter analysis revealed that apparently several rhizobial glb genes are not regulated by a -10 promoter but might be regulated by -35 and Fnr (fumarate-nitrate reduction regulator)-like promoters. Mapping analysis revealed that rhizobial fhbs and thbs are flanked by a variety of genes whereas several rhizobial sdgbs and gcss are flanked by genes coding for proteins involved in the metabolism of nitrates and nitrites and chemotaxis, respectively. Phenetic analysis showed that rhizobial Glbs segregate into the M, S and T Glb families, while structural analysis showed that predicted rhizobial SDgbs and fHbs and GCSs globin domain and tHbs fold into the 3/3- and 2/2-folding, respectively. Spectra from B. japonicum USDA38 and USDA58 soluble extracts exhibited peaks and troughs characteristic of bacterial and vertebrate Glbs thus indicating that putative Glbs are synthesized in B. japonicum USDA38 and USDA58.

A bioinformatics insight to rhizobial globins: gene identification and mapping, polypeptide sequence and phenetic analysis, and protein modeling.

Globins (Glbs) are proteins widely distributed in organisms. Three evolutionary families have been identified in Glbs: the M, S and T Glb families. The M Glbs include flavohemoglobins (fHbs) and single-domain Glbs (SDgbs); the S Glbs include globin-coupled sensors (GCSs), protoglobins and sensor single domain globins, and the T Glbs include truncated Glbs (tHbs). Structurally, the M and S Glbs exhibit 3/3-folding whereas the T Glbs exhibit 2/2-folding. Glbs are widespread in bacteria, including several rhizobial genomes. However, only few rhizobial Glbs have been characterized. Hence, we characterized Glbs from 62 rhizobial genomes using bioinformatics methods such as data mining in databases, sequence alignment, phenogram construction and protein modeling. Also, we analyzed soluble extracts from Bradyrhizobium japonicum USDA38 and USDA58 by (reduced + carbon monoxide (CO) minus reduced) differential spectroscopy. Database searching showed that only fhb, sdgb, gcs and thb genes exist in the rhizobia analyzed in this work. Promoter analysis revealed that apparently several rhizobial glb genes are not regulated by a -10 promoter but might be regulated by -35 and Fnr (fumarate-nitrate reduction regulator)-like promoters. Mapping analysis revealed that rhizobial fhbs and thbs are flanked by a variety of genes whereas several rhizobial sdgbs and gcss are flanked by genes coding for proteins involved in the metabolism of nitrates and nitrites and chemotaxis, respectively. Phenetic analysis showed that rhizobial Glbs segregate into the M, S and T Glb families, while structural analysis showed that predicted rhizobial SDgbs and fHbs and GCSs globin domain and tHbs fold into the 3/3- and 2/2-folding, respectively. Spectra from B. japonicum USDA38 and USDA58 soluble extracts exhibited peaks and troughs characteristic of bacterial and vertebrate Glbs thus indicating that putative Glbs are synthesized in B. japonicum USDA38 and USDA58.

Lipid-modified azurin of Neisseria meningitidis is a copper protein localized on the outer membrane surface and not regulated by FNR

The laz gene of Neisseria meningitidis is predicted to encode a lipid-modified azurin (Laz). Laz is very similar to azurin, a periplasmic protein, which belongs to the copper-containing proteins in the cupredoxin superfamily. In other bacteria, azurin is an electron donor to nitrite reductase, an important enzyme in the denitrifying process. It is not known whether Laz could function as an electron transfer protein in this important pathogen. Laz protein was heterologously expressed in Escherichia coli and purified. Electrospray mass spectrometry indicated that the Laz protein contains one copper ion. Laz was shown to be redox-active in the presence of its redox center copper ion. When oxidized, Laz exhibits an intense blue colour and absorbs visible light around 626nm. The absorption is lost when exposed to diethyldithiocarbamate, a copper chelating agent. Polyclonal antibodies were raised against purified Laz for detecting expression of Laz under different growth conditions and to determine the orientation of Laz on the outer membrane. The expression of Laz under microaerobic and microaerobic denitrifying conditions was slightly higher than that under aerobic conditions. However, the expression of Laz was similar between the wild type strain and an fnr mutant, suggesting that Fumarate/Nitrate reduction regulator (FNR) does not regulate the expression of Laz despite the presence of a partial FNR box upstream of the laz gene. We propose that some Laz protein is exposed on the outer membrane surface of N. meningitidis as the αLaz antibodies can increase killing by complement in a capsule deficient N. meningitidis strain, in a dose-dependent fashion.

Prediction of the Tertiary Structure and the Dimerization Interface of FNR Protein in <i>Klebsiella pneumoniae</i>

FNR (fumarate nitrate reduction regulator) is an important oxygen regulation protein, which plays a major role in altering gene expression between aerobic and anaerobic conditions. In this study, the tertiary structure of FNR protein in Klebsiella pneumoniae was established using homology modelling. The model was modified and optimized using molecular mechanics and molecular dynamics simulations. In addition, Support Vector Machines (SVMs) was applied to predict the possible binding sites patches of FNR. The residues conservations were further analyzed and it was found that the Asn138-Ser162 region of FNR protein was the putative dimerization interface.

Characterization of Salmonella enterica serovar Typhimurium aconitase A

Aconitases (Acn) are iron-sulfur proteins that catalyse the reversible isomerization of citrate and isocitrate via the intermediate cis-aconitate in the Krebs cycle. Some Acn proteins are bi-functional and under conditions of iron starvation and oxidative stress lose their iron-sulfur clusters and become post-transcriptional regulators by binding specific mRNA targets. Many bacterial species possess two genetically distinct aconitase proteins, AcnA and AcnB. Current understanding of the regulation and functions of AcnA and AcnB in dual Acn bacteria is based on a model developed in Escherichia coli. Thus, AcnB is the major Krebs cycle enzyme expressed during exponential growth, whereas AcnA is a more stable, stationary phase and stress-induced enzyme, and both E. coli Acns are bi-functional. Here a second dual Acn bacterium, Salmonella enterica serovar Typhimurium (S. Typhimurium), has been analysed. Phenotypic traits of S. Typhimurium acn mutants were consistent with AcnB acting as the major Acn protein. Promoter fusion experiments indicated that acnB transcription was ~10-fold greater than that of acnA and that acnA expression was regulated by the cyclic-AMP receptor protein (CRP, glucose starvation), the fumarate nitrate reduction regulator (FNR, oxygen starvation), the ferric uptake regulator (Fur, iron starvation) and the superoxide response protein (SoxR, oxidative stress). In contrast to E. coli, S. Typhimurium acnA was not induced in the stationary phase. Furthermore, acnA expression was enhanced in an acnB mutant, presumably to partially compensate for the lack of AcnB activity. Isolated S. Typhimurium AcnA protein had kinetic and mRNA-binding properties similar to those described for E. coli AcnA. Thus, the work reported here provides a second example of the regulation and function of AcnA and AcnB proteins in a dual Acn bacterium.

X-ray snapshots of possible intermediates in the time course of synthesis and degradation of protein-bound Fe 4 S 4 clusters

Fe4S4 clusters are very common versatile prosthetic groups in proteins. Their redox property of being sensitive to O2-induced oxidative damage is, for instance, used by the cell to sense oxygen levels and switch between aerobic and anaerobic metabolisms, as exemplified by the fumarate, nitrate reduction regulator (FNR). Using the hydrogenase maturase HydE from Thermotoga maritima as a template, we obtained several unusual forms of FeS clusters, some of which are associated with important structural changes. These structures represent intermediate states relevant to both FeS cluster assembly and degradation. We observe one Fe2S2 cluster bound by two cysteine persulfide residues. This observation lends structural support to a very recent Raman study, which reported that Fe4S4-to-Fe2S2 cluster conversion upon oxygen exposure in FNR resulted in concomitant production of cysteine persulfide as cluster ligands. Similar persulfide ligands have been observed in vitro for several other Fe4S4 cluster-containing proteins. We have also monitored FeS cluster conversion directly in our protein crystals. Our structures indicate that the Fe4S4-to-Fe2S2 change requires large structural modifications, which are most likely responsible for the dimer-monomer transition in FNR.

Physical organization and phylogenetic analysis of acdR as leucine-responsive regulator of the 1-aminocyclopropane-1-carboxylate deaminase gene acdS in phytobeneficial Azospirillum lipoferum 4B and other Proteobacteria

The phytostimulatory alphaproteobacterium Azospirillum lipoferum 4B exhibits the plant-beneficial gene acdS, which enables deamination of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Here, we show that acdS is in the vicinity of acdR, a homolog to leucine-responsive regulator lrp, in A. lipoferum 4B and most other acdS+ Proteobacteria. Unlike in Beta- and Gammaproteobacteria, acdS (and acdR) is preferentially located on symbiotic islands and plasmids in Alphaproteobacteria. In A. lipoferum 4B, acdS was mapped on a 750-kb plasmid that is lost during phenotypic variation, whereas other phytobeneficial genes such as nifH (associative nitrogen fixation) are maintained. In Proteobacteria, the phylogenies of acdR and acdS were largely but not totally congruent, despite physical proximity of the genes, regardless of whether DNA or deduced protein sequences were used. Potential Lrp, cAMP receptor protein (CRP) and fumarate-nitrate reduction regulator (FNR) binding sites were evidenced in the acdS promoter regions of strain 4B and most of 46 other acdS+ Proteobacteria. Indeed, transcriptional and enzymatic analyses done in vitro pointed to the involvement of Lrp- and FNR-like transcriptional up-regulation of ACC deaminase activity in A. lipoferum 4B. This is the first synteny, phylogenetic, and functional analysis of factors modulating acdS expression in Azospirillum plant growth-promoting rhizobacterium.

Se(VI) Reduction and the Precipitation of Se(0) by the Facultative Bacterium Enterobacter cloacae SLD1a-1 Are Regulated by FNR

The fate of selenium in the environment is controlled, in part, by microbial selenium oxyanion reduction and Se(0) precipitation. In this study, we identified a genetic regulator that controls selenate reductase activity in the Se-reducing bacterium Enterobacter cloacae SLD1a-1. Heterologous expression of the global anaerobic regulatory gene fnr (fumarate nitrate reduction regulator) from E. cloacae in the non-Se-reducing strain Escherichia coli S17-1 activated the ability to reduce Se(VI) and precipitate insoluble Se(0) particles. Se(VI) reduction by E. coli S17-1 containing the fnr gene occurred at rates similar to those for E. cloacae, with first-order reaction constants of k = 2.07 x 10(-2) h(-1) and k = 3.36 x 10(-2) h(-1), respectively, and produced elemental selenium particles with identical morphologies and short-range atomic orders. Mutation of the fnr gene in E. cloacae SLD1a-1 resulted in derivative strains that were deficient in selenate reductase activity and unable to precipitate elemental selenium. Complementation by the wild-type fnr sequence restored the ability of mutant strains to reduce Se(VI). Our findings suggest that Se(VI) reduction and the precipitation of Se(0) by facultative anaerobes are regulated by oxygen-sensing transcription factors and occur under suboxic conditions.

Overlapping promoters modulate Fnr- and ArcA-dependent anaerobic transcriptional activation of the focApfl operon in Escherichia coli.

The recently identified P6A promoter of the anaerobically inducible focApfl operon of Escherichia coll overlaps the Fnr (fumarate-nitrate reduction regulator)-dependent P6 promoter. The Fnr-binding site of P6 and the -35 hexamer sequence of P6A are shared between the promoters. Inactivation of P6A, through introduction of a -10 hexamer mutation, resulted in enhanced anaerobic induction of operon expression. The dependence on the ArcA (aerobic respiration control regulator) and Fnr transcription factors for anaerobic induction was tested for several focA-lacZ and pfl-lacZ gene fusions. Anaerobic induction became more dependent on Fnr in derivatives lacking a functional P6A promoter compared with wild-type constructs. Moreover, aerobic expression of the focA gene was reduced by the p6A mutation, as was the dependence on ArcA for anaerobic induction. Inactivation of P6 severely reduced Fnr-dependent anaerobic induction, in accord with previous findings. Transcription analyses demonstrated that a mutation in the -10 hexamer sequence of either P6A or P6 did not adversely affect transcription from the remaining promoter. Taken together, these results indicate that the P6A promoter moderates the Fnr-dependent activation of P6 through competition for RNA polymerase binding.