Fur Gene Research Articles

Deciphering the Molecular Mechanism of Peracetic Acid Response in Listeria monocytogenes

Peracetic acid (PAA), a strong oxidizing agent, has been widely used as a disinfectant in food processing settings as it does not produce harmful chlorinated by-products. In the present study, the transcriptional response of Listeria monocytogenes to a sub-lethal concentration of PAA (2.5 ppm) was assessed using RNA-sequencing (RNA-seq). Our analysis revealed 12 differentially expressed protein-coding genes, of which nine were upregulated (ohrR, ohrA, rpsN, lmo0637, lmo1973, fur, lmo2492, zurM, and lmo1007), and three were down-regulated (argG, lmo0604 and lmo2156) in PAA-treated samples compared to the control samples. A non-coding small RNA gene (rli32) was also found to be down-regulated. In detail, the organic peroxide toxicity protection (OhrA-OhrR) system, the metal homeostasis genes fur and zurM, the SbrE-regulated lmo0636-lmo0637 operon and a carbohydrate phosphotransferase system (PTS) operon component were induced under exposure of L. monocytogenes to PAA. Hence, this study identified key elements involved in the primary response of L. monocytogenes to oxidative stress caused by PAA, including the expression of the peroxide detoxification system and fine-tuning the levels of redox-active metals in the cell. The investigation of the molecular mechanism of PAA response in L. monocytogenes is of utmost importance for the food industry, as residual PAA can lead to stress tolerance in pathogens.

Regulatory mechanism of C4-dicarboxylates in cyclo (Phe-Pro) production

Cyclo (Phe-Pro) (cFP), a cyclic dipeptide with notable antifungal, antibacterial, and antiviral properties, shows great promise for biological control of plant diseases. Produced as a byproduct by non-ribosomal peptide synthetases (NRPS), the regulatory mechanism of cFP biosynthesis remains unclear. In a screening test of 997 Tn5 mutants of Burkholderia seminalis strain R456, we identified eight mutants with enhanced antagonistic effects against Fusarium graminearum (Fg). Among these, mutant 88’s culture filtrate contained cFP, confirmed through HPLC and LC-MS, which actively inhibited Fg. The gene disrupted in mutant 88 is part of the Dct transport system (Dct-A, -B, -D), responsible for C4-dicarboxylate transport. Knockout mutants of Dct genes exhibited higher cFP levels than the wild type, whereas complementary strains showed no significant difference. Additionally, the presence of exogenous C4-dicarboxylates reduced cFP production in wild type R456, indicating that these substrates negatively regulate cFP synthesis. Given that cFP synthesis is related to NRPS, we previously identified an NRPS cluster in R456, horizontally transferred from algae. Specifically, knocking out gene 2061 within this NRPS cluster significantly reduced cFP production. A Fur box binding site was predicted upstream of gene 2061, and yeast one-hybrid assays confirmed Fur protein binding, which increased with additional C4-dicarboxylates. Knockout of the Fur gene led to up-regulation of gene 2061 and increased cFP production, suggesting that C4-dicarboxylates suppress cFP synthesis by enhancing Fur-mediated repression of gene 2061.

Ferric uptake regulator (Fur) in Vibrio mimicus acts as an activator of citrate cycle and oxidative phosphorylation pathways, while enhancing virulence potential

Ferric uptake regulator (Fur) is a crucial bacterial regulator that controls the uptake of iron by bacteria, hence influencing their survival and pathogenicity. Vibrio mimicus is an epidemic pathogen that is harmful to aquatic animals and human health, with the characteristics of rapid onset and high mortality after infecting the fish. At present, the function and mechanism of Fur in V. mimicus are still unclear. In this study, the fur gene deletion strain and complementation strain of V. mimicus were constructed. Following fur deletion, proteomic analysis revealed that the abundance of 258 proteins was considerably up-regulated while the abundance of 307 proteins was dramatically down-regulated. The primary roles of these differentially expressed proteins were in a variety of pathways, including virulence, metabolism, enzymes, and so on. Among them, citrate cycle and oxidative phosphorylation were the main pathways affected by Fur. The activities of key enzymes in these two pathways were assessed. The results indicated a decrease in the activities of respiratory chain complexes II and IV, succinate dehydrogenase (SDH), and α-ketoglutarate dehydrogenase (α-KGDH) in the Δfur strain. Conversely, the activity of isocitrate dehydrogenase (NAD-IDH) was increased. Phenotypic characterization showed that the Δfur strain exhibited delayed growth, reduced motility, defective biofilm formation, decreased resistance to oxidative stress, increased adhesion, and decreased virulence. Besides, the LD50 of the Δfur strain was 31-fold higher than that of the WT strain. These results indicate that Fur serves as an activator for the citrate cycle and oxidative phosphorylation pathway in V. mimicus, and contributing to its virulence. Our findings enhance the understanding of the biological functions of Fur and lay the groundwork for further investigation into the regulatory mechanisms of Fur in V. mimicus.

The transcriptional regulator Fur modulates the expression of uge, a gene essential for the core lipopolysaccharide biosynthesis in Klebsiella pneumoniae

BackgroundKlebsiella pneumoniae is a Gram-negative pathogen that has become a threat to public health worldwide due to the emergence of hypervirulent and multidrug-resistant strains. Cell-surface components, such as polysaccharide capsules, fimbriae, and lipopolysaccharides (LPS), are among the major virulence factors for K. pneumoniae. One of the genes involved in LPS biosynthesis is the uge gene, which encodes the uridine diphosphate galacturonate 4-epimerase enzyme. Although essential for the LPS formation in K. pneumoniae, little is known about the mechanisms that regulate the expression of uge. Ferric uptake regulator (Fur) is an iron-responsive transcription factor that modulates the expression of capsular and fimbrial genes, but its role in LPS expression has not yet been identified. This work aimed to investigate the role of the Fur regulator in the expression of the K. pneumoniae uge gene and to determine whether the production of LPS by K. pneumoniae is modulated by the iron levels available to the bacterium.ResultsUsing bioinformatic analyses, a Fur-binding site was identified on the promoter region of the uge gene; this binding site was validated experimentally through Fur Titration Assay (FURTA) and DNA Electrophoretic Mobility Shift Assay (EMSA) techniques. RT-qPCR analyses were used to evaluate the expression of uge according to the iron levels available to the bacterium. The iron-rich condition led to a down-regulation of uge, while the iron-restricted condition resulted in up-regulation. In addition, LPS was extracted and quantified on K. pneumoniae cells subjected to iron-replete and iron-limited conditions. The iron-limited condition increased the amount of LPS produced by K. pneumoniae. Finally, the expression levels of uge and the amount of the LPS were evaluated on a K. pneumoniae strain mutant for the fur gene. Compared to the wild-type, the strain with the fur gene knocked out presented a lower LPS amount and an unchanged expression of uge, regardless of the iron levels.ConclusionsHere, we show that iron deprivation led the K. pneumoniae cells to produce higher amount of LPS and that the Fur regulator modulates the expression of uge, a gene essential for LPS biosynthesis. Thus, our results indicate that iron availability modulates the LPS biosynthesis in K. pneumoniae through a Fur-dependent mechanism.

Functional characterization of Fur from the strict anaerobe Clostridioides difficile provides insight into its redox-driven regulatory capacity.

Clostridioides (formerly Clostridium) difficile is a leading cause of infectious diarrhea associated with antibiotic therapy. The ability of this anaerobic pathogen to acquire enough iron to proliferate under iron limitation conditions imposed by the host largely determines its pathogenicity. However, since high intracellular iron catalyzes formation of deleterious reactive hydroxyl radicals, iron uptake is tightly regulated at the transcriptional level by the ferric uptake regulator Fur. Several studies relate lacking a functional fur gene in C. difficile cells to higher oxidative stress sensitivity, colonization defect and less toxigenicity, although Fur does not appear to directly regulate either oxidative stress response genes or pathogenesis genes. In this work, we report the functional characterization of C. difficile Fur and describe an additional oxidation sensing Fur-mediated mechanism independent of iron, which affects Fur DNA-binding. Using electrophoretic mobility shift assays, we show that Fur binding to the promoters of fur, feoA and fldX genes, identified as iron and Fur-regulated genes in vivo, is specific and does not require co-regulator metal under reducing conditions. Fur treatment with H2O2 produces dose-dependent soluble high molecular weight species unable to bind to target promoters. Moreover, Fur oligomers are dithiotreitol sensitive, highlighting the importance of some interchain disulfide bond(s) for Fur oligomerization, and hence for activity. Additionally, the physiological electron transport chain NADPH-thioredoxin reductase/thioredoxin from Escherichia coli reduces inactive oligomerized C. difficile Fur that recovers activity. In conjunction with available transcriptomic data, these results suggest a previously underappreciated complexity in the control of some members of the Fur regulon that is based on Fur redox properties and might be fundamental for the adaptive response of C. difficile during infection.

Regulation of Cysteine Homeostasis and Its Effect on Escherichia coli Sensitivity to Ciprofloxacin in LB Medium.

Cysteine and its derivatives, including H2S, can influence bacterial virulence and sensitivity to antibiotics. In minimal sulfate media, H2S is generated under stress to prevent excess cysteine and, together with incorporation into glutathione and export into the medium, is a mechanism of cysteine homeostasis. Here, we studied the features of cysteine homeostasis in LB medium, where the main source of sulfur is cystine, whose import can create excess cysteine inside cells. We used mutants in the mechanisms of cysteine homeostasis and a set of microbiological and biochemical methods, including the real-time monitoring of sulfide and oxygen, the determination of cysteine and glutathione (GSH), and the expression of the Fur, OxyR, and SOS regulons genes. During normal growth, the parental strain generated H2S when switching respiration to another substrate. The mutations affected the onset time, the intensity and duration of H2S production, cysteine and glutathione levels, bacterial growth and respiration rates, and the induction of defense systems. Exposure to chloramphenicol and high doses of ciprofloxacin increased cysteine content and GSH synthesis. A high inverse relationship between log CFU/mL and bacterial growth rate before ciprofloxacin addition was revealed. The study points to the important role of maintaining cysteine homeostasis during normal growth and antibiotic exposure in LB medium.

Impact of the Iron-Response Regulator Genes on the Release of Iron and Aluminum from Biotite by Rhizobium pusense S41

In this study, the biotite weathering activities of strain S41 and mutants with deletion of the iron response regulator genes fur, rirA, and irr were compared. S41ΔrirA had a 35%–60% increase in Fe and Al concentrations, a 95%–112% increase in siderophore production, and a 18%–22% increase in EPS concentration, while S41Δirr had a 29%–48% decrease in Fe and Al concentrations and a 32%–42% decrease in siderophore production, compared with strain S41. Under iron-sufficient conditions, S41ΔrirA had a 1.5-fold–2.7-fold increase in siderophore production, while there was similar siderophore production between strain S41 and S41Δirr. Under iron-limited conditions, S41Δirr had a 34%–40% decrease in siderophore production compared with strain S41. The expression of genes related to the synthesis of siderophores, iron uptake and transporters, and EPS production in strain S41 significantly increased in iron-limited conditions compared with that in iron-sufficient conditions. The expression of genes associated with the synthesis of siderophores and iron uptake and transporters significantly increased in S41ΔrirA in iron-sufficient and iron-limited conditions but decreased in S41Δirr in iron-limited conditions. Our results suggested that rirA- and irr-modulated multiple genes may be related to the mineral weathering activities of strain S41.

Δfur mutant as a potential live attenuated vaccine (LAV) candidate protects American eels (Anguilla rostrata) from Vibrio harveyi infection

The eel farming industry is highly susceptible to Vibriosis. Although various types of vaccines against Vibriosis have been investigated, there is limited research on decreasing the virulence of Vibrions through gene knockout and utilizing it as live attenuated vaccines (LAV). In this study, we aim to develop a LAV candidate against Vibrio harveyi infection in American eels (Anguilla rostrata) using a ferric uptake regulator (fur) gene mutant strain of V. harveyi (Δfur mutant). After the eels were administrated with the Δfur mutant at the dose of 4 × 102 cfu/g body weight, the phagocytic activity of the leucocytes, plasma IgM antibody titers, activity of lysozyme and Superoxide Dismutase (SOD) enzyme, and gene expression levels of 18 immune related proteins were detected to evaluate the protection effect of the LAV. Preliminary findings suggest that the LAV achieved over 60% relative percent survival (RPS) after the American eels were challenged by a wild-type strain of V. harveyi infection on 28 and 42 days post the immunization (dpi). The protection was mainly attributed to increased plasma IgM antibody titers, higher levels of lysozyme, enhanced activity of SOD and some regulated genes encoded immune related proteins. Together, the Δfur mutant strain of V. harveyi, as a novel LAV vaccine, demonstrates promising protective effects against V. harveyi infection in American eels, thus presenting a potential candidate vaccine for fish farming.

Comparative phenotype and transcriptome analysis revealed the role of ferric uptake regulator (Fur) in the virulence of Vibrio harveyi isolated from diseased American eel (Anguilla rostrata).

Vibrio harveyi is commonly found in salt and brackish water and is recognized as a serious bacterial pathogen in aquaculture worldwide. In this study, we cloned the ferric uptake regulator (fur) gene from V. harveyi wild-type strain HA_1, which was isolated from diseased American eels (Anguilla rostrata) and has a length of 450 bp, encoding 149 amino acids. Then, a mutant strain, HA_1-Δfur, was constructed through homologous recombination of a suicide plasmid (pCVD442). The HA_1-Δfur mutant exhibited weaker biofilm formation and swarming motility, and 18-fold decrease (5.5%) in virulence to the American eels; compared to the wild-type strain, the mutant strain showed time and diameter differences in growth and haemolysis, respectively. Additionally, the adhesion ability of the mutant strain was significantly decreased. Moreover, there were 15 different biochemical indicators observed between the two strains. Transcriptome analysis revealed that 875 genes were differentially expressed in the Δfur mutant, with 385 up-regulated and 490 down-regulated DEGs. GO and KEGG enrichment analysis revealed that, compared to the wild-type strain, the type II and type VI secretion systems (T2SS and T6SS), amino acid synthesis and transport and energy metabolism pathways were significantly down-regulated, but the ABC transporters and biosynthesis of siderophore group non-ribosomal peptides pathways were up-regulated in the Δfur strain. The qRT-PCR results further confirmed that DEGs responsible for amino acid transport and energy metabolism were positively regulated, but DEGs involved in iron acquisition were negatively regulated in the Δfur strain. These findings suggest that the virulence of the Δfur strain was significantly decreased, which is closely related to phenotype changing and gene transcript regulation.

Mechanisms of nanoscale zero-valent iron mediating aerobic denitrification in Pseudomonas stutzeri by promoting electron transfer and gene expression

Aerobic denitrification and its mechanism by P. stutzeri was investigated in the presence of nanoscale zero-valent iron (nZVI). The removal of nitrate and ammonia was accelerated and the nitrite nitrogen accumulation was reduced by nZVI. The particle size and dosage of nZVI were key factors for enhancing aerobic denitrification. nZVI reduced the negative effects of low carbon/nitrogen, heavy metals, surfactants and salts to aerobic denitrification. nZVI and its dissolved irons were adsorbed into the bacteria cells, enhancing the transfer of electrons from nicotinamide adenine dinucleotide (NADH) to nitrate reductase. Moreover, the activities of NADH-ubiquinone reductase involved in the respiratory system, and the denitrifying enzymes were increased. The expression of denitrifying enzyme genes napA and nirS, as well as the iron metabolism gene fur, were promoted in the presence of nZVI. This work provides a strategy for enhancing the biological denitrification of wastewater using the bio-stimulation of nanomaterials.

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.

Role of Catecholamine Hormone to Increase the Virulence of Aeromonas hydrophila

Extreme environmental conditions have been widely reported to trigger stress in exposed living organisms, and such conditions affect their homeostatics. Sress conditions lead vertebrates to release stress hormones, such as catecholamine Norepinephrin (NE) and Dopamine (Dopamine), which involve the hypothalamus-sympathetic-chromaffin (HSC) axis and the hypothalamus-pituitary-interrenal axis (HPI axis). These two axis systems produce catecholamine hormones (including adrenaline, noradrenaline, and dopamine) and corticosteroid hormones (especially cortisol). The presence of these two groups of hormones in the circulatory system of the hosts can act as a signal for pathogenic bacteria, such as Aeromonas hydrophilla commonly found in aquaculture, to start their infection process. These two types of hormones play a role in the expression of Aeromonas hydrophila genes encoding formation of flagella (allowing bacterial cells to swim rapidly to their host), pilli, toxin production, and activation of biofilm formation process. The stress conditions experienced by fishes (which triggers the secretion of catecholamine hormones) can significantly reduce the fish's immune system to function, and this will be exploited by Aeromonas hydrophila to increase its metabolic rate, growth, and virulence in such stressed fish. Specifically, catecholamine hormones can also act as a signal for the Aeromonas hydrophila to activate the ferric uptake regulator gene (fur gene), allowing this pathogenic bacterium to optimally chelate ions of iron (Fe3+) from its environment, resulting in the excessive growth of the pathogen and an increase in its pathogenicity.

Coevolution of furA-Regulated Hyper-Inflammation and Mycobacterial Resistance to Oxidative Killing through Adaptation to Hydrogen Peroxide.

Mycobacterium tuberculosis (Mtb) is highly resistant to host oxidative killing. We hypothesized that the evolutionary adaptation of M. smegmatis to hydrogen peroxide (H2O2) would endow the nonpathogenic Mycobacterium persistent in a host. In the study, we screened a highly H2O2-resistant strain (mc2114) via evolutionary H2O2 adaptation in vitro. The MIC of mc2114 to H2O2 is 320 times that of wild-type mc2155. Mouse infection experiments showed that mc2114, similar to Mtb, was persistent in the lungs and caused high lethality in mice with restricted responses of NOX2, ROS, IFN-γ, decreased macrophage apoptosis, and overexpressed inflammatory cytokines in the lungs. Whole-genome sequencing analysis revealed that mc2114 harbored 29 single nucleotide polymorphisms in multiple genes; one of them was on the furA gene that caused FurA deficiency-mediated overexpression of KatG, a catalase-peroxidase to detoxify ROS. Complementation of mc2114 with a wild-type furA gene reversed lethality and hyper-inflammatory response in mice with rescued overexpression of KatG and inflammatory cytokines, whereas NOX2, ROS, IFN-γ, and macrophage apoptosis remained reduced. The results indicate that although FurA regulates KatG expression, it does not contribute significantly to the restriction of ROS response. Instead, FurA deficiency is responsible for the detrimental pulmonary inflammation that contributes to the severity of the infection, a previously nonrecognized function of FurA in mycobacterial pathogenesis. The study also indicates that mycobacterial resistance to oxidative burst results from complex mechanisms involving adaptive genetic changes in multiple genes. IMPORTANCE Mycobacterium tuberculosis (Mtb) causes human tuberculosis (TB), which has killed more people in human history than any other microorganism. However, the mechanisms underlying Mtb pathogenesis and related genes have not yet been fully elucidated, which impedes the development of effective strategies for containing and eradicating TB. In the study, we generated a mutant of M. smegmatis (mc2114) with multiple mutations by an adaptive evolutionary screen with H2O2. One of the mutations in furA caused a deficiency of FurA, which mediated severe inflammatory lung injury and higher lethality in mice by overexpression of inflammatory cytokines. Our results indicate that FurA-regulated pulmonary inflammation plays a critical role in mycobacterial pathogenesis in addition to the known downregulation of NOX2, ROS, IFN-γ responses, and macrophage apoptosis. Further analysis of the mutations in mc2114 would identify more genes related to the increased pathogenicity and help in devising new strategies for containing and eradicating TB.

Regulation of iron metabolism is critical for the survival of Salmonella Typhimurium in pasteurized milk

Salmonella is known to survive in raw/pasteurized milk and cause foodborne outbreaks. Lactoferrin, present in milk from all animal sources, is an iron-binding glycoprotein that limits the availability of iron to pathogenic bacteria. Despite the presence of lactoferrins, Salmonella can grow in milk obtained from different animal sources. However, the mechanism by which Salmonella overcomes iron scarcity induced by lactoferrin in milk is not evaluated yet. Salmonella employs the DNA binding transcriptional regulator Fur (ferric update regulator) to mediate iron uptake during survival in iron deplete conditions. To understand the importance of Fur in Salmonella milk growth, we profiled the growth of Salmonella Typhimurium Δfur (ST4/74Δfur) in both bovine and camel milk. ST4/74Δfur was highly inhibited in milk compared to wild-type ST4/74, confirming the importance of Fur mediated regulation of iron metabolism in Salmonella milk growth. We further studied the biology of ST4/74Δfur to understand the importance of iron metabolism in Salmonella milk survival. Using increasing concentrations of FeCl3, and the antibiotic streptonigrin we show that iron accumulates in the cytoplasm of ST4/74Δfur. We hypothesized that the accumulated iron could activate oxidative stress via Fenton’s reaction leading to growth inhibition. However, the inhibition of ST4/74Δfur in milk was not due to Fenton’s reaction, but due to the ‘iron scarce’ conditions of milk and microaerophilic incubation conditions which made the presence of the fur gene indispensable for Salmonella milk growth. Subsequently, survival studies of 14 other transcriptional mutants of ST4/74 in milk confirmed that RpoE-mediated response to extracytoplasmic stress is also important for the survival of Salmonella in milk. Though we have data only for fur and rpoE, many other Salmonella transcriptional factors could play important roles in the growth of Salmonella in milk, a theme for future research on Salmonella milk biology. Nevertheless, our data provide early insights into the biology of milk-associated Salmonella.

The C-terminal domain of the ferric uptake regulator (Fur) binds a [2Fe-2S] cluster to sense the intracellular free iron content in Escherichia coli.

Escherichia coli ferric uptake regulator (Fur) binds a [2Fe-2S] cluster, not a mononuclear iron, when the intracellular free iron content is elevated in E. coli cells. Here we report that the C-terminal domain (residues 83-148) of E. coli Fur (Fur-CTD) is sufficient to bind the [2Fe-2S] cluster in response to elevation of the intracellular free iron content in E. coli cells. Deletion of gene fur in E. coli cells increases the intracellular free iron content and promotes the [2Fe-2S] cluster binding in the Fur-CTD in the cells grown in LB medium under aerobic growth conditions. When the Fur-CTD is expressed in wild type E. coli cells grown in M9 medium supplemented with increasing concentrations of iron, the Fur-CTD also progressively binds a [2Fe-2S] cluster with a maximum occupancy of about 36%. Like the E. coli Fur-CTD, the CTD of the Haemophilus influenzae Fur can also bind a [2Fe-2S] cluster in wild type E. coli cells grown in M9 medium supplemented with increasing concentrations of iron, indicating that binding of the [2Fe-2S] cluster in the C-terminal domain is highly conserved among Fur proteins. The results suggest that the Fur-CTD can be used as a physiological probe to assess the intracellular free iron content in bacteria.

Vibrio splendidus Fur regulates virulence gene expression, swarming motility, and biofilm formation, affecting its pathogenicity in Apostichopus japonicus.

Vibrio splendidus is an opportunistic pathogen that causes skin ulcer syndrome and results in huge losses to the Apostichopus japonicus breeding industry. Ferric uptake regulator (Fur) is a global transcription factor that affects varieties of virulence-related functions in pathogenic bacteria. However, the role of the V. splendidus fur (Vsfur) gene in the pathogenesis of V. splendidus remains unclear. Hence, we constructed a Vsfur knock-down mutant of the V. splendidus strain (MTVs) to investigate the role of the gene in the effect of biofilm, swarming motility, and virulence on A. japonicus. The result showed that the growth curves of the wild-type V. splendidus strain (WTVs) and MTVs were almost consistent. Compared with WTVs, the significant increases in the transcription of the virulence-related gene Vshppd mRNA were 3.54- and 7.33-fold in MTVs at the OD600 of 1.0 and 1.5, respectively. Similarly, compared with WTVs, the significant increases in the transcription of Vsm mRNA were 2.10- and 15.92-fold in MTVs at the OD600 of 1.0 and 1.5, respectively. On the contrary, the mRNA level of the flagellum assembly gene Vsflic was downregulated 0.56-fold in MTVs at the OD600 of 1.0 compared with the WTVs. MTVs caused delayed disease onset time and reduced A. japonicus mortality. The median lethal doses of WTVs and MTVs were 9.116 × 106 and 1.658 × 1011 CFU·ml-1, respectively. Compared with WTVs, the colonization abilities of MTVs to the muscle, intestine, tentacle, and coelomic fluid of A. japonicus were significantly reduced. Correspondingly, the swarming motility and biofilm formation in normal and iron-replete conditions were remarkably decreased compared with those of WTVs. Overall, these results demonstrate that Vsfur contributes to the pathogenesis of V. splendidus by regulating virulence-related gene expression and affecting its swarming and biofilm formation abilities.

Transcriptomic analysis of the effect of deferoxamine exposure on the growth, photosynthetic activity and iron transfer of Microcystis aeruginosa

Deferoxamine (DFB) is a trihydroxamic acid siderophore that chelates with iron (Fe) to form iron-siderophore complexes. The existence of siderophores in nature changes the form of iron and affects the absorption and utilization of iron by organisms. However, the relationship between siderophores and the growth of Cyanobacteria is largely unknown. In this study, the cellular and transcriptomic responses to the addition of DFB were investigated. A high concentration of DFB (12 mg/L) significantly inhibited the growth of Cyanobacteria cells, reduced photosynthetic activity, and induced the production of peroxidase, with the highest inhibition rate of algal growth of 74.82%. These indexes were also affected for the low (3 mg/L) and medium concentration (6 mg/L) groups, but this difference is closely related to the growth stage of Cyanobacteria cells. This may be due to competition between the cell-associated iron-binding part/system and the extracellular Fe (Ⅲ)-DFB ligand. Transcriptome results showed that most of the genes involved in iron uptake and transport were down-regulated, and only the fur gene encoding the iron uptake regulator protein was significantly up-regulated. Most genes related to photosynthesis, glycolysis, and fatty acid metabolism were also down-regulated, while the obvious up-regulation of a few genes may be a complex regulation in response to the down-regulation of most genes. These findings will provide important insights into the effects of siderophores on iron bioavailability in algae.

Lactobacilli Downregulate Transcription Factors in Helicobacter pylori That Affect Motility, Acid Tolerance and Antimicrobial Peptide Survival

Helicobacter pylori infection triggers inflammation that may lead to gastritis, stomach ulcers and cancer. Probiotic bacteria, such as Lactobacillus, have been of interest as treatment options, however, little is known about the molecular mechanisms of Lactobacillus-mediated inhibition of H. pylori pathogenesis. In this work, we investigated the effect of Lactobacillus culture supernatants, so-called conditioned medium (CM), from two gastric isolates, L. gasseri and L. oris, on the expression of transcriptional regulators in H. pylori. Among the four known two-component systems (TCSs), i.e., ArsRS, FlgRS, CheAY and CrdRS, the flagellar regulator gene flgR and the acid resistance associated arsS gene were down-regulated by L. gasseri CM, whereas expression of the other TCS-genes remained unaffected. L. gasseri CM also reduced the motility of H. pylori, which is in line with reduced flgR expression. Furthermore, among six transcription factors of H. pylori only the ferric uptake regulator gene fur was regulated by L. gasseri CM. Deletion of fur further led to dramatically increased sensitivity to the antimicrobial peptide LL-37. Taken together, the results highlight that released/secreted factors of some lactobacilli, but not all, downregulate transcriptional regulators involved in motility, acid tolerance and LL-37 sensitivity of H. pylori.

Detecção dos genes Fur, AmoA e pvcAB em Aeromonas hydrophila isoladas de organismos aquáticos e o impacto da presença desses genes sobre o crescimento bacteriano com diferentes concentrações de ferro

ABSTRACT Infection caused by Aeromonas brings great harm to fish farming. Among the factors associated with bacterial pathogenesis, iron uptake can contribute to the survival and virulence of bacteria within hosts. The aim of this study was to check the presence of genes related to iron uptake in Aeromonas hydrophila deriving from aquatic organisms in the São Francisco Valley and associate the presence of these genes with the ability to grow in media containing different concentrations of iron. The DNAs of 41 isolates were extracted and used in PCRs to verify the presence of the Fur, AmoA and pvcAB genes related to iron uptake. The growth of the isolates belonging to different genetic profiles was verified in culture media containing different iron concentrations. Two isolates were positive for the presence of the Fur gene, seven for the AmoA gene and two for the pvcAB gene. The growth test showed that the low availability of iron did not interfere in the growth of the isolates, nor in the isolate that did not contain any of the genes evaluated in this study, suggesting that the iron uptake’s mechanisms of the tested isolates may be related to other genes and proteins.

The role of urease in the acid stress response and fimbriae expression in Klebsiella pneumoniae CG43.

Two urease operons were identified in Klebsiella pneumoniae CG43, ure-1 and ure-2. This study investigates whether a differential regulation of the expression of ure-1 and ure-2 exists and how urease activity influences the acid stress response and expression of type 1 and type 3 fimbriae. The ureA1 and ureA2 gene specific deletion mutants were constructed. Promoter activity was assessed using a LacZ reporter system. The sensitivity to acid stress was determined by assessing the survival after pH 2.5 treatment. The influence on type 1 and type 3 fimbriae expression was assessed using western blotting and mannose-sensitive yeast agglutination and biofilm formation assay, respectively. Bacterial growth analysis in mM9-U or modified Stuart broth revealed that ure-1 was the principal urease system, and ure-2 had a negative effect on ure-1 activity. Deletion of the fur or nac gene had no apparent effect on the activity of Pure1, Pure2-1, and Pure2-2. The Pure2-2 activity was enhanced by deletion of the hns gene. ureA1 deletion increased acid stress sensitivity, whereas the deleting effect of ureA2 was notable without hns. Deletion of ureA1 or ureA2 significantly induced the expression of type 1 fimbriae but decreased MrkA production and biofilm formation. ure-1 is the primary expression system in K. pneumoniae CG43, while ure-2 is active in the absence of hns. Impairment of urease activity increases the sensitivity to acid stress, and the accumulation of urea induces the expression of type 1 fimbriae but represses type 3 fimbriae expression.