Fur Mutant Research Articles

The Staphylococcus aureus small non-coding RNA IsrR regulates TCA cycle activity and virulence.

Staphylococcus aureus has evolved mechanisms to cope with low iron (Fe) availability in host tissues. S. aureus uses the ferric uptake transcriptional regulator (Fur) to sense titers of cytosolic Fe. Upon Fe depletion, apo-Fur relieves transcriptional repression of genes utilized for Fe uptake. We demonstrate that an S. aureus Δfur mutant has decreased expression of acnA, which codes for the Fe-dependent enzyme aconitase. Decreased acnA expression prevented the Δfur mutant from growing with amino acids as sole carbon and energy sources. Suppressor analysis determined that a mutation in isrR, which produces a regulatory RNA, permitted growth by decreasing isrR transcription. The decreased AcnA activity of the Δfur mutant was partially relieved by an ΔisrR mutation. Directed mutation of bases predicted to facilitate the interaction between the acnA transcript and IsrR, decreased the ability of IsrR to control acnA expression in vivo and IsrR bound to the acnA transcript in vitro. IsrR also bound to the transcripts coding the alternate TCA cycle proteins sdhC, mqo, citZ, and citM. Whole cell metal analyses suggest that IsrR promotes Fe uptake and increases intracellular Fe not ligated by macromolecules. Lastly, we determined that Fur and IsrR promote infection using murine skin and acute pneumonia models.

Efficient markerless genetic manipulation of Pasteurella multocida using lacZ and pheSm as selection markers.

Pasteurella multocida is a zoonotic conditional pathogen that infects multiple livestock species, causing substantial economic losses in the animal husbandry industry. An efficient markerless method for gene manipulation may facilitate the investigations of P. multocida gene function and pathogenesis of P. multocida. Herein, a temperature-sensitive shuttle vector was constructed using lacZ as a selection marker, and markerless glgB, opa, and hyaE mutants of P. multocida were subsequently constructed through blue-white colony screening. The screening efficiency of markerless deletion strains was improved by the lacZ system, and the method could be used for multiple gene deletions. However, the fur mutant was unavailable via this method. Therefore, we constructed a pheSm screening system based on mutated phenylalanine tRNA synthetase as a counterselection marker to achieve fur deletion mutant. The transformed strain was sensitive to 20 mM p-chloro-phenylalanine, demonstrating the feasibility of pheSm as a counter-selective marker. The pheSm system was used for markerless deletions of glgB, opa, and hyaE as well as fur that could not be screened by the lacZ system. A comparison of screening efficiencies of the system showed that the pheSm counterselection system was more efficient than the lacZ system and broadly applicable for mutant screening. The methods developed herein may provide valuable tools for genetic manipulation of P. multocida.IMPORTANCEPasteurella multocida is a highly contagious zoonotic pathogen. An understanding of its underlying pathogenic mechanisms is of considerable importance and requires efficient species-specific genetic tools. Herein, we propose a screening system for P. multocida mutants using lacZ or pheSm screening markers. We evaluated the efficiencies of both systems, which were used to achieve markerless deletion of multiple genes. The results of this study support the use of lacZ or pheSm as counterselection markers to improve counterselection efficiency in P. multocida. This study provides an effective genetic tool for investigations of the virulence gene functions and pathogenic mechanisms of P. multocida.

Ferric uptake regulator (fur) affects the pathogenicity of Aeromonas veronii TH0426 by regulating flagellar assembly and biofilm formation

Aeromonas veronii has serious implications for food safety and the aquaculture industry. The iron uptake system can effectively regulate the life activities and pathogenicity of microorganisms, and Fur is one of the key proteins. In this experiment, we constructed the Δ fur mutant strain of A. veronii TH0426 using the pRE112 suicide plasmid and the C- fur complementary strain using the pBBR1-MCS generalized host expression plasmid to research the effects of fur gene on the biological function and mechanisms of virulence regulation of TH0426. It was found that when the fur gene was deleted, the polar flagella of TH0426 was disrupted and the biofilm formation ability was significantly reduced, with consequent changes in motility, flagellation-related gene expression, drug resistance, and tolerance. Transcriptome analysis of TH0426 wild-type and Δ fur mutant showed that fur gene could directly or indirectly regulate bacterial flagella assembly, antibiotic resistance, growth and metabolism. In the pathogenic test, compared with the TH0426 wild-type, Δ fur significantly reduced the cytotoxicity to EPC cells at 30 min, 1 h and 2 h, reduced the adhesion and invasion ability to EPC cells by 2.38-fold (P < 0.001), increased LD50 of zebrafish by 58.33-fold (P < 0.001) and decreased bacterial load at 12 h, 24 h, and 48 h. Meanwhile, the result of C- fur was higher than that of Δ fur and lower than that of the TH0426 wild-type in all experiments. In summary, the fur gene is essential for flagellar assembly and biofilm formation and is able to indirectly affect some of the biological properties and animal pathogenicity of TH0426. This research helps to explain the function of the fur gene of the iron uptake system and its role in the pathogenesis of animal.

Ferric uptake regulator (Fur) binds a [2Fe-2S] cluster to regulate intracellular iron homeostasis in Escherichia coli

Intracellular iron homeostasis in bacteria is primarily regulated by ferric uptake regulator (Fur). It has been postulated that when intracellular free iron content is elevated, Fur binds ferrous iron to downregulate the genes for iron uptake. However, the iron-bound Fur had not been identified in any bacteria until we recently found that Escherichia coli Fur binds a [2Fe-2S] cluster, but not a mononuclear iron, in E.coli mutant cells that hyperaccumulate intracellular free iron. Here, we report that E.coli Fur also binds a [2Fe-2S] cluster in wildtype E.coli cells grown in M9 medium supplemented with increasing concentrations of iron under aerobic growth conditions. Additionally, we find that binding of the [2Fe-2S] cluster in Fur turns on its binding activity for specific DNA sequences known as the Fur-box and that removal of the [2Fe-2S] cluster from Fur eliminates its Fur-box binding activity. Mutation of the conserved cysteine residues Cys-93 and Cys-96 to Ala in Fur results in the Fur mutants that fail to bind the [2Fe-2S] cluster, have a diminished binding activity for the Fur-box invitro, and are inactive to complement the function of Fur invivo. Our results suggest that Fur binds a [2Fe-2S] cluster to regulate intracellular iron homeostasis in response to elevation of intracellular free iron content in E.coli cells.

Evaluation of the immunoprotection efficacy of Riemerella anatipestifer fur-deficient mutant as an attenuated vaccine

Riemerella anatipestifer (R. anatipestifer, RA) is an infectious pathogen that causes septicemia and polyserositis in ducks. Our previous studies showed that RA CH-1 ∆fur was significantly attenuated in ducklings, which highlights the potential of this strain as a live attenuated vaccine. In this study, it was shown that infection with 109 CFU of the fur mutant did not cause any clinical symptoms or significant histological lesions in 3-day-old ducklings and that the bacteria were readily cleared by the host within 3 d. Compared with the nonvaccinated group, the group inoculated with the mutant strain RA CH-1 ∆fur exhibited protection of ducklings against a high-dose (2.28×1010 CFU) challenge with the wild-type strain RA CH-1. Moreover, the average body weights and body weight gains of the Δfur-inoculated group were not significantly affected by the challenge. Further analysis revealed that RA CH-1 ∆fur elicited higher IgY titers and that the serum antibody levels persisted for at least 49 d after immunization. Overall, our study showed that RA CH-1 ∆fur is a safe and effective vaccine candidate that is expected to play an important role in RA CH-1 infection prevention in the duck industry.

The sbiTRS Operon Contributes to Stenobactin-Mediated Iron Utilization in Stenotrophomonas maltophilia.

Iron is an essential micronutrient for various bacterial cellular processes. Fur is a global transcriptional regulator participating in iron homeostasis. Stenotrophomonas maltophilia is a ubiquitous environmental bacterium that has emerged as an opportunistic pathogen. To elucidate the novel regulatory mechanism behind iron homeostasis in S. maltophilia, wild-type KJ and KJΔFur, a fur mutant, were subjected to transcriptome assay. A five-gene cluster, sbiBA-sbiTRS, was significantly upregulated in KJΔFur. SbiAB is an ATP type efflux pump, SbiT is an inner membrane protein, and SbiSR is a two-component regulatory system (TCS). The sbiTRS operon organization was verified by reverse transcription-PCR (RT-PCR). Localization prediction and bacterial two-hybrid studies revealed that SbiT resided in the inner membrane and had an intramembrane interaction with SbiS. In iron-replete conditions, SbiT interacted with SbiS and maintained SbiSR TCS in a resting state. In response to iron depletion stress, SbiT no longer interacted with SbiS, leading to SbiSR TCS activation. The iron source utilization assay demonstrated the contribution of SbiSR TCS to stenobactin-mediated ferric iron utilization but notto the utilization of hemin and ferric citrate. Furthermore, SmeDEF and SbiAB pumps, known stenobactin secretion outlets, were members of the SbiSR regulon. Collectively, in an iron-depleted condition, SbiSR activation is regulated by Fur at the transcriptional level and by SbiT at the posttranslational level. Activated SbiSR contributes to stenobactin-mediated ferric iron utilization by upregulating the smeDEF and sbiAB operons. SbiSR is the first TCS found to be involved in iron homeostasis in S. maltophilia. IMPORTANCE Therapeutic options for Stenotrophomonas maltophilia infections are limited because S. maltophilia is intrinsically resistant to several antibiotics. Iron is an essential element for viability, but iron overload is a lethal threat to bacteria. Therefore, disruption of iron homeostasis can be an alternative strategy to cope with S. maltophilia infection. The intricate regulatory networks involved in iron hemostasis have been reported in various pathogens; however, little is known about S. maltophilia. Herein, a novel sbiTRS operon, a member of Fur regulon, was characterized. SbiT, an inner membrane protein, negatively modulated the SbiSR two-component regulatory system by intramembrane protein-protein interaction with SbiS. In response to iron-depleted stress, SbiSR was activated via the regulation of Fur and SbiT. Activated SbiSR upregulated smeDEF and sbiAB, which contributed to stenobactin-mediated ferric iron utilization. A novel fur-sbiT-sbiSR-smeDEF/sbiAB regulatory circuit in S. maltophilia was revealed.

A putative siderophore receptor of Gallibacterium anatis 12656-12 under Fur control also binds hemoglobin.

Pasteurellaceae family members obtain iron directly from host proteins or through siderophore-dependent mechanisms. Although Gallibacterum anatis expresses different virulence factors, its response to growth under iron restriction is unknown. G. anatis cultured in the presence of 2,2′-dipyridyl, up-expressed an approximately 65 kDa protein and repressed the expression of a 70 kDa protein. MALDI-TOF analysis of those proteins indicated homology with CirA (65 kDa), a protein involved in iron-siderophore acquisition in Mannheimia succinoproducens and a TonB-dependent receptor (70 kDa protein), a protein that binds chicken hemoglobin; however, G. anatis siderophore production was not detected by chromo azurol S (CAS)-BHI agar determination. This putative G. anatis siderophore receptor is under Fur control, but not the hemoglobin binding protein, as observed in G. anatis 12656-12 fur mutant (Ω fur 126.13) grown in the presence or not of 2,2′-dipyridyl. The addition of FeCl3 to the culture medium diminished the growth and biofilm production in approximately 30% and 35%, respectively, in the wild-type strain, but the growth of Ω fur 126.13 strain was not affected and biofilm production increased in 35%. G. anatis Ω fur 126.13 presented lower virulence when it was inoculated to 35-day-old chickens in comparison to the wild-type strain. The induction of more than one iron uptake mechanism could benefit pathogenic microorganisms such as Gallibacterium.

Conditional growth defect of Bordetella pertussis and Bordetella bronchiseptica ferric uptake regulator (fur) mutants.

Outer-membrane vesicles (OMVs) are promising tools in the development of novel vaccines against the respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica. Unfortunately, vesiculation by bordetellae is too low for cost-effective vaccine production. In other bacteria, iron limitation or inactivation of the fur gene has been shown to increase OMV production, presumably by downregulation of the mla genes, which encode machinery for maintenance of lipid asymmetry in the outer membrane. Here, we followed a similar approach in bordetellae. Whereas a fur mutant was readily obtained in B. bronchiseptica, a B. pertussis fur mutant could only be obtained in iron-deplete conditions, indicating that a fur mutation is conditionally lethal in this bacterium. The fur mutants displayed a growth defect in iron-replete media, presumably because constitutive expression of iron-uptake systems resulted in iron intoxication. Accordingly, expression of the Escherichia coli ferritin FtnA to sequester intracellularly accumulated iron rescued the growth of the mutants in these media. The fur mutations led to the constitutive expression of novel vaccine candidates, such as the TonB-dependent receptors FauA for the siderophore alcaligin and BhuR for heme. However, neither inactivation of fur nor growth under iron limitation improved vesiculation, presumably because the expression of the mla genes appeared unaffected.

Involvement of the hemP-hemA-smlt0796-smlt0797 Operon in Hemin Acquisition by Stenotrophomonas maltophilia.

ABSTRACTThe hemin acquisition system of Stenotrophomonas maltophilia was elucidated in this study. To identify the TonB-dependent outer membrane receptor for hemin in S. maltophilia, the hemin acquisition systems of Pseudomonas aeruginosa were referenced. PhuR, HasA, and HxuA are three known TonB-dependent outer membrane receptors involved in hemin acquisition by P. aeruginosa. Thus, HemA (Smlt0795) and Smlt2937, the orthologs of PhuR and HasA/HxuA in S. maltophilia, were first considered. KJΔEnt, a stenobactin-null strain, was used as the parental strain for the hemin utilization assay. Deletion of hemA, but not Smlt2937, of KJΔEnt impaired hemin acquisition under iron-depleted conditions, indicating that HemA is the TonB-dependent receptor for hemin uptake. The hemA gene is a member of the hemP-hemA-smlt0796-smlt0797 operon, whose expression was upregulated in a fur mutant and under iron-depleted conditions. The contribution of the hemP-hemA-smlt0796-smlt0797 operon to hemin acquisition was investigated by in-frame deletion mutant construction and hemin utilization assays. Inactivation of hemP, smlt0796, and smlt0797 of KJΔEnt insignificantly affected hemin acquisition under iron-depleted conditions. However, hemP deletion in a fur mutant increased hemin acquisition under iron-depleted conditions. Collectively, we revealed that (i) HemA likely functions as the outer membrane receptor for hemin uptake; (ii) HemP, a predicted transcriptional factor, apparently functions as a repressor of the expression of the hemA transcript; and (iii) in a fur mutant, HemP has a negative impact on hemin acquisition under iron-depleted conditions.IMPORTANCE Stenotrophomonas maltophilia is an emerging multidrug-resistant opportunistic pathogen, increasing the difficulty of treatment of this infection. Iron is a critical element for bacterial viability. Heme is the most abundant iron source in the human host; thus, heme is the major iron source for a pathogen in the infection niche. Blocking iron acquisition from heme can be an alternative strategy to control S. maltophilia infection. Although several hemin acquisition systems have been reported in various pathogens, very little is known about the hemin acquisition systems of S. maltophilia. By in-frame deletion mutant construction and hemin utilization assays, we demonstrated that HemA (Smlt0795) is the TonB-dependent outer membrane receptor for hemin uptake and that HemP (Smlt0794), a predicted transcriptional factor, had a negative impact on hemin acquisition in a fur mutant. The negative regulatory role of HemP in hemin acquisition is first reported.

Ferric Uptake Regulator (Fur) Regulates Intracellular Iron Homeostasis via reversible binding of a [2Fe‐2S] cluster in Escherichia coli

It has generally been assumed that when intracellular “free” iron content is elevated in bacteria, a global transcription factor Fur (Ferric uptake regulator) binds Fe(II) to regulate expression of its target genes and achieve intracellular iron homeostasis. Fur is highly conserved among bacteria, and purified Fur proteins from different bacteria may exist as a homodimer or tetramer with each monomer containing three metal binding sites. While two of the metal binding sites (site 1 and site 2) in purified Fur proteins are often occupied by Zn(II), the iron‐bound Fur has never been isolated from any bacteria. Here we report that Escherichia coli Fur binds a [2Fe‐2S] cluster at site 3 (via Cys‐93 and Cys‐96) in the E. coli mutant cells in which intracellular “free” iron content is elevated due to deficiency of iron‐sulfur cluster biogenesis. Depletion of intracellular “free” iron content effectively removes the [2Fe‐2S] cluster from Fur, indicating that binding of a [2Fe‐2S] cluster in Fur is determined by intracellular “free” iron content. Fur homolog from Haemophilus influenzae also binds a [2Fe‐2S] cluster at site 3 when expressed in E. coli cells, suggesting that binding of a [2Fe‐2S] cluster in Fur is highly conserved. While the Zn(II)‐bound E. coli Fur has a basal binding activity for its target DNA sequence known as Fur‐box, binding of a [2Fe‐2S] cluster significantly enhances its Fur‐box binding activity. Similarly, binding of a [2Fe‐2S] cluster is required for the strong binding activity of H. influenzaeFur for Fur‐box in vitro. Mutation of Cys‐93 to Ala results in an E. coli Fur mutant that fails to bind the [2Fe‐2S] cluster, loses the enhanced binding activity for Fur‐box, and is inactive to complement the physiological function of E. coli Fur in E. coli cells. Our results suggest that E. coli Fur and H. influenzae Fur binds a [2Fe‐2S] cluster to regulate intracellular iron homeostasis in response to elevation of intracellular “free” iron content in cells.

Negatively Regulated Aerobactin and Desferrioxamine E by Fur in Pantoea ananatis Are Required for Full Siderophore Production and Antibacterial Activity, but Not for Virulence.

Pantoea ananatis is an emerging plant pathogen that causes disease in economically important crops such as rice, corn, onion, melon, and pineapple, and it also infects humans and insects. In this study, we identified biosynthetic gene clusters of aerobactin and desferrioxamine E (DFO-E) siderophores by using the complete genome of P. ananatis PA13 isolated from rice sheath rot. P. ananatis PA13 exhibited the strongest antibacterial activity against Erwinia amylovora and Yersinia enterocolitica (Enterobacterales). Mutants of aerobactin or DFO-E maintained antibacterial activity against E. amylovora and Y. enterocolitica, as well as in a siderophore activity assay. However, double aerobactin and DFO-E gene deletion mutants completely lost siderophore and antibacterial activity. These results reveal that both siderophore biosynthetic gene clusters are essential for siderophore production and antibacterial activity in P. ananatis PA13. A ferric uptake regulator protein (Fur) mutant exhibited a significant increase in siderophore production, and a Fur-overexpressing strain completely lost antibacterial activity. Expression of the iucA, dfoJ, and foxA genes was significantly increased in the Δfur mutant background, and expression of these genes returned to wild-type levels after fur compensation. These results indicate that Fur negatively regulates aerobactin and DFO-E siderophores. However, siderophore production was not required for P. ananatis virulence in plants, but it appears to be involved in the microbial ecology surrounding the plant environment. This study is the first to report the regulation and functional characteristics of siderophore biosynthetic genes in P. ananatis. IMPORTANCE Pantoea ananatis is a bacterium that causes diseases in several economically important crops, as well as in insects and humans. This bacterium has been studied extensively as a potentially dangerous pathogen due to its saprophytic ability. Recently, the types, biosynthetic gene clusters, and origin of the siderophores in the Pantoea genus were determined by using genome comparative analyses. However, few genetic studies have investigated the characteristics and functions of siderophores in P. ananatis. The results of this study revealed that the production of aerobactin and desferrioxamine E in the rice pathogen P. ananatis PA13 is negatively regulated by Fur and that these siderophores are essential for antibacterial activity against Erwinia amylovora and Yersinia enterocolitica (Enterobacterales). However, siderophore production was not required for P. ananatis virulence in plants, but it appears to be involved in the microbial ecology surrounding the plant environment.

Functional characterization of Fur in iron metabolism, oxidative stress resistance and virulence of Riemerella anatipestifer

Iron is essential for most bacteria to survive, but excessive iron leads to damage by the Fenton reaction. Therefore, the concentration of intracellular free iron must be strictly controlled in bacteria. Riemerella anatipestifer (R. anatipestifer), a Gram-negative bacterium, encodes the iron uptake system. However, the iron homeostasis mechanism remains largely unknown. In this study, it was shown that compared with the wild type R. anatipestifer CH-1, R. anatipestifer CH-1Δfur was more sensitive to streptonigrin, and this effect was alleviated when the bacteria were cultured in iron-depleted medium, suggesting that the fur mutant led to excess iron accumulation inside cells. Similarly, compared with R. anatipestifer CH-1∆recA, R. anatipestifer CH-1∆recAΔfur was more sensitive to H2O2-induced oxidative stress when the bacteria were grown in iron-rich medium rather than iron-depleted medium. Accordingly, it was shown that R. anatipestifer CH-1∆recAΔfur produced more intracellular ROS than R. anatipestifer CH-1∆recA in iron-rich medium. Electrophoretic mobility shift assays showed that R. anatipestifer CH-1 Fur suppressed the transcription of putative iron uptake genes through binding to their promoter regions. Finally, it was shown that compared with the wild type, R. anatipestifer CH-1Δfur was significantly attenuated in ducklings and that the colonization ability of R. anatipestifer CH-1Δfur in various tissues or organs was decreased. All these results suggested that Fur is important for iron homeostasis in R. anatipestifer and its pathogenic mechanism.

Free Rather Than Total Iron Content Is Critically Linked to the Fur Physiology in Shewanella oneidensis.

Ferric uptake regulator (Fur) is a transcriptional regulator playing a central role in iron homeostasis of many bacteria, and Fur inactivation commonly results in pleiotropic phenotypes. In Shewanella oneidensis, a representative of dissimilatory metal-reducing γ-proteobacteria capable of respiring a variety of chemicals as electron acceptors (EAs), Fur loss substantially impairs respiration. However, to date the mechanism underlying the physiological phenomenon remains obscure. This investigation reveals that Fur loss compromises activity of iron proteins requiring biosynthetic processes for their iron cofactors, heme in particular. We then show that S. oneidensis Fur is critical for maintaining heme homeostasis by affecting both its biosynthesis and decomposition of the molecule. Intriguingly, the abundance of iron-containing proteins controlled by H2O2-responding regulator OxyR increases in the fur mutant because the Fur loss activates OxyR. By comparing suppression of membrane-impermeable, membrane-permeable, and intracellular-only iron chelators on heme deficiency and elevated H2O2 resistance, our data suggest that the elevation of the free iron content by the Fur loss is likely to be the predominant factor for the Fur physiology. Overall, these results provide circumstantial evidence that Fur inactivation disturbs bacterial iron homeostasis by altering transcription of its regulon members, through which many physiological processes, such as respiration and oxidative stress response, are transformed.

Pathogenicity and immunogenicity of Edwardsiella piscicida ferric uptake regulator (fur) mutations in zebrafish

Edwardsiella piscicida is the etiological agent of edwardsiellosis in fish and causes severe economic losses in global aquaculture. Vaccination would be the most effective method to prevent infectious diseases and their associated economic losses. The ferric uptake regulator (Fur) is an important transcriptional global regulator of Gram-negative bacteria. In this study, we examined the regulatory function of Fur in E. piscicida. We designed a strain that displays features of the wild-type virulent strain of E. piscicida at the time of immunization to enable strains first to effectively colonize lymphoid tissues and then to exhibit a regulated delayed attenuation in vivo to preclude inducing disease symptoms. Regulated delayed attenuation in vivo is based on the substitution of a tightly regulated araC ParaBAD cassette for the promoter of the fur gene such that expression of this gene is dependent on arabinose provided during growth. Thus, following E. piscicida mutant colonization of lymphoid tissues, the Fur protein ceases to be synthesized due to the absence of arabinose such that attenuation is gradually manifest in vivo to preclude induction of diseases symptoms. We deleted the promoter, including all sequences that interact with activator or repressor proteins, for the fur gene, and substituted the improved araC ParaBAD cassette to yield an E. piscicida strain with the ΔPfur170:TT araC ParaBADfur deletion-insertion mutation (χ16012). Compared to the wild-type strain J118, χ16012 exhibited retarded growth and enhanced siderophore production in the absence of arabinose. mRNA levels of Fur-regulated genes were analyzed in iron deplete or replete condition in wild-type and fur mutant strains. We observed zebrafish immunized with χ16012 showed better colonization and protection compared to the Δfur (χ16001). Studies showed that E. piscicida strain χ16012 is attenuated and induces systemic and mucosal IgM titer in zebrafish. In addition, we found an increase in transcript levels of tnf-α, il-1β, il-8 and ifn-γ in different tissues of zebrafish immunized with χ16012 compared to the unimmunized group. We conclude that, E. piscicida with regulated delayed attenuation could be an effective immersion vaccine for the aquaculture industry.

Rsp activates expression of the Cnt system in Staphylococcus aureus

BackgroundThe Cnt system is crucial for the optimal import of essential metals in metal-limiting conditions and contributes to virulence in S. aureus. In a screen for regulators of efflux pumps in a phage-based ultra-high-density transposon library, we identified Rsp as a candidate regulator of the cntE gene.ResultsA two-fold decrease in expression of all genes of the cnt operon was observed by RT-qPCR in the rsp mutant compared to the parental strain, indicating that Rsp acts as an activator of the cnt operon. To determine whether the Rsp activation depends on iron, we compared mutant and parent cnt expression under varying metal conditions. A 2-fold reduction in cnt gene expression was detected in the rsp mutant in TSB, and a slightly smaller decrease (1.9, 1.7, and 1.5-fold changes for cntK, cmtA, and cntE respectively) was observed after addition of dipyridyl. The greatest decrease was seen with addition of FeSO4 (4.1, 5.3 and 6.3-fold changes for cntK, cmtA and cntE respectively). These findings suggest that Rsp activates the cnt operon in low and high iron conditions. To study the relationship between Rsp and the cnt repressors Fur and Zur, we created single and double mutants. Both fur and zur single mutants had significant increases in cnt gene expression compared to the parental strain, as did the fur rsp double mutant. The zur rsp double mutant also had a significant increase in cntK expression and a trend in increases in cntA and cntE expression just below statistical significance. Thus, the ability of Fur and Zur to repress cnt gene expression are not eliminated by the presence of Rsp. However, there were significantly smaller increases in cnt gene expression in the double mutants compared to single mutants, suggesting that Rsp activation can still occur in the absence of these repressors. To determine if Rsp directly modulates expression of cnt genes, incubation of purified Rsp caused a DNA-specific band shift for the cntK and cntA promoters.ConclusionsRsp activation may act to maintain basal cellular levels of staphylopine to scavenge free metals when needed, in addition to metal dependent regulation by Fur and Zur.

Biochemical characterization of ferric uptake regulator (Fur) from Aliivibrio salmonicida. Mapping the DNA sequence specificity through binding studies and structural modelling

Iron is an essential nutrient for bacteria, however its propensity to form toxic hydroxyl radicals at high intracellular concentrations, requires its acquisition to be tightly regulated. Ferric uptake regulator (Fur) is a metal-dependent DNA-binding protein that acts as a transcriptional regulator in maintaining iron metabolism in bacteria and is a highly interesting target in the design of new antibacterial drugs. Fur mutants have been shown to exhibit decreased virulence in infection models. The protein interacts specifically with DNA at binding sites designated as ‘Fur boxes’. In the present study, we have investigated the interaction between Fur from the fish pathogen Aliivibrio salmonicida (AsFur) and its target DNA using a combination of biochemical and in silico methods. A series of target DNA oligomers were designed based on analyses of Fur boxes from other species, and affinities assessed using electrophoretic mobility shift assay. Binding strengths were interpreted in the context of homology models of AsFur to gain molecular-level insight into binding specificity.

Vibrio parahaemolyticus FcrX, a Fur-controlled regulator that inhibits repression by Fur.

Iron is an essential nutrient for most organisms, but its limited availability and inherent toxicity necessitate the strict regulation of iron homeostasis. In bacteria, iron starvation affects a broad range of phenotypes including virulence, motility and biofilm formation. For Vibrio parahaemolyticus, a marine bacterium and pathogen, iron limitation is a signal modulating swarmer cell differentiation. In this work, we show the iron regulation of swarming works through the ferric uptake regulator protein Fur. We identified a new Fur-controlled regulator that is upregulated upon iron starvation. FcrX is a 144-amino acid protein containing a domain of unknown function (DUF2753) with three tetratricopeptide repeats. We found that overexpressing fcrX+ was sufficient to induce swarming, luminescence and iron uptake gene expression in multiple Vibrio species; furthermore, ectopic expression increased the transcription of a Fur-controlled gene in Escherichia coli. FcrX production increased intracellular iron. Thus, the overexpression of fcrX+ phenocopied a fur mutant and may prove a generally useful tool to ectopically derepress the Fur regulon. Both V. parahaemolyticus and E. coli Fur interacted with FcrX, and this interaction was altered by iron availability. These data support a model in which this new regulator of iron homeostasis limits the repressive action of Fur.

Transcriptional response to metal starvation in the emerging pathogen Mycoplasma genitalium is mediated by Fur-dependent and –independent regulatory pathways

ABSTRACT Transition metals participate in numerous enzymatic reactions and they are essential for survival in all living organisms. For this reason, bacterial pathogens have evolved dedicated machineries to effectively compete with their hosts and scavenge metals at the site of infection. In this study, we investigated the mechanisms controlling metal acquisition in the emerging human pathogen Mycoplasma genitalium. We observed a robust transcriptional response to metal starvation, and many genes coding for predicted lipoproteins and ABC-transporters were significantly up-regulated. Transcriptional analysis of a mutant strain lacking a metalloregulator of the Fur family revealed the activation of a full operon encoding a putative metal transporter system and a gene coding for a Histidine-rich lipoprotein (Hrl). We recognized a conserved sequence with dyad symmetry within the promoter region of the Fur-regulated genes. Mutagenesis of the predicted Fur operator within the hrl promoter abrogated Fur- and metal-dependent expression of a reporter gene. Metal starvation still impelled a strong transcriptional response in the fur mutant, demonstrating the existence of Fur-independent regulatory pathways controlling metal homeostasis. Finally, analysis of metal accumulation in the wild-type strain and the fur mutant by ICP-MS revealed an important role of Fur in nickel acquisition.

New evolving strategies revealed by transcriptomic analysis of a fur- mutant of the cyanotrophic bacterium Pseudomonas pseudoalcaligenes CECT 5344.

SummaryThe transcriptomic analysis (RNA‐seq) of a fur mutant of P. pseudoalcaligenes CECT 5344 has revealed that Fur regulates the expression of more than 100 genes in this bacterial strain, most of them negatively. The highest upregulated genes in response to fur deletion, with respect to the wild type, both cultivated in LB medium, corresponded to genes implicated in iron uptake. They include both TonB‐dependent siderophore transporters for the active transport across the outer membrane, and ABC‐type and MSF‐type transporters for the active transport across the cytoplasmic membrane. Therefore, the main response of this bacterium to iron limitation is expressing genes necessary for metabolism of Fe siderophores produced by other microorganisms (xenosiderophores). The number of genes whose expression decreased in the fur− mutant, as well as its normalized expression (fold change), was lower. Among them, it is remarkable the presence of one of the two cas operons of the two CRISP/Cas clusters was detected in the genome of this bacterium. The transcriptome was validated by qPCR, including the decrease in the expression of cas genes (cse1). The expression of cse1 was also decreased by limiting the amount of iron, carbon or nitrogen in the medium, or by adding menadione, a compound that causes oxidative stress. The higher decrease in cse1 expression was triggered by the addition of cyanide in minimal medium. These results suggest that this bacterium responds to stress conditions, and especially to cyanide, taking a reasonable risk with respect to both the uptake of (TonB‐dependent receptors gates) and the tolerance to (reduced immunity) foreign nucleic acids. In conjunction, this can be considered a yet unknown molecular mechanism forcing bacterial evolution.

Identification of Fur in Pasteurella multocida and the Potential of Its Mutant as an Attenuated Live Vaccine.

Pasteurella multocida is a pathogenic microorganism that causes a variety of serious diseases in humans and animals worldwide. The global regulator gene, fur, plays an important role in pathogenesis and regulates the virulence of many bacteria. Here, we identified a fur gene in P. multocida by complementing a Salmonella Choleraesuis Δfur mutant, and characterized a fur mutant strain of P. multocida. The P. multocida Δfur mutant strain exhibited no significant differences in growth and outer membrane protein (OMP) profiles when the complemented strain was compared to the parent. Ducks were used as the model organism to determine the virulence and protection efficacy induced by Δfur mutant strain. Animal experiments showed that colonization by the mutant was decreased by oral infection of live Δfur mutant strain. The LD50 of the ducks infected with the Δfur mutant was 146-fold higher than that of the ducks infected with the wild-type strain when administered through the oral route. Evaluation of the immunogenicity and protective efficacy of the Δfur mutant of P. multocida revealed strong serum IgY and bile IgA immune responses following oral inoculation with the Δfur strain. Ducks that were orally inoculated with the Δfur mutant strain demonstrated 62% protection efficacy against severe lethal challenge with the wild-type P. multocida. This study provides new insights into P. multocida virulence and the potential use of an attenuated vaccine against P. multocida.