Regulation Of Siderophores Research Articles

A Disturbed Siderophore Transport Inhibits Myxobacterial Predation

Understanding the intrinsic mechanisms of bacterial competition is a fundamental question. Iron is an essential trace nutrient that bacteria compete for. The most prevalent manner for iron scavenging is through the secretion of siderophores. Although tremendous efforts have focused on elucidating the molecular mechanisms of siderophores biosynthesis, export, uptake, and regulation of siderophores, the ecological aspects of siderophore-mediated competition are not well understood. We performed predation and bacterial competition assays to investigate the function of siderophore transport on myxobacterial predation. Deletion of msuB, which encodes an iron chelate uptake ABC transporter family permease subunit, led to a reduction in myxobacterial predation and intracellular iron, but iron deficiency was not the predominant reason for the decrease in the predation ability of the ∆msuB mutant. We further confirmed that obstruction of siderophore transport decreased myxobacterial predation by investigating the function of a non-ribosomal peptide synthetase for siderophore biosynthesis, a TonB-dependent receptor, and a siderophore binding protein in M. xanthus. Our results showed that the obstruction of siderophores transport decreased myxobacterial predation ability through the downregulation of lytic enzyme genes, especially outer membrane vesicle (OMV)-specific proteins. This work provides insight into the mechanism of siderophore-mediated competition in myxobacteria.

Metatranscriptomic Analysis Reveals Active Bacterial Communities in Diabetic Foot Infections.

Despite the extended view of the composition of diabetic foot infections (DFIs), little is known about which transcriptionally active bacterial communities are pertinent to infection, and if any differences are associated with increased infection severity. We applied a RNA sequencing approach to analyze the composition, function, and pathogenicity of the active bacterial communities in DFIs. Taxonomic profiling of bacterial transcripts revealed the presence of 14 bacterial phyla in DFIs. The abundance of the Spiroplasma, Vibrio, and Mycoplasma were significantly different in different infection severities (P < 0.05). Mild and severe stages of infections were dominated by Staphylococcus aureus and Porphyromonas asaccharolytica, respectively. A total of 132 metabolic pathways were identified of which ribosome and thiamin being among the most highly transcribed pathways. Moreover, a total of 131 antibiotic resistance genes, primarily involved in the multidrug efflux pumps/exporters, were identified. Furthermore, iron acquisition systems (synthesize and regulation of siderophores) and pathways involved in the synthesis and regulation of cell-surface components associated with adhesion, colonization, and movement of bacterial cells were the most common virulence factors. These virulence factors may help bacteria compete for scares resources and survive the host wound proteases. Characterization of transcriptionally active bacterial communities can help to provide an understanding of the role of key pathogens in the development of DFIs. Such information can be clinically useful allowing replacement of DFIs empirical therapy with targeted treatment.

YPR2 is a regulator of light modulated carbon and secondary metabolism in Trichoderma reesei

BackgroundFilamentous fungi have evolved to succeed in nature by efficient growth and degradation of substrates, but also due to the production of secondary metabolites including mycotoxins. For Trichoderma reesei, as a biotechnological workhorse for homologous and heterologous protein production, secondary metabolite secretion is of particular importance for industrial application. Recent studies revealed an interconnected regulation of enzyme gene expression and carbon metabolism with secondary metabolism.ResultsHere, we investigated gene regulation by YPR2, one out of two transcription factors located within the SOR cluster of T. reesei, which is involved in biosynthesis of sorbicillinoids. Transcriptome analysis showed that YPR2 exerts its major function in constant darkness upon growth on cellulose. Targets (direct and indirect) of YPR2 overlap with induction specific genes as well as with targets of the carbon catabolite repressor CRE1 and a considerable proportion is regulated by photoreceptors as well. Functional category analysis revealed both effects on carbon metabolism and secondary metabolism. Further, we found indications for an involvement of YPR2 in regulation of siderophores. In agreement with transcriptome data, mass spectrometric analyses revealed a broad alteration in metabolite patterns in ∆ypr2. Additionally, YPR2 positively influenced alamethicin levels along with transcript levels of the alamethicin synthase tex1 and is essential for production of orsellinic acid in darkness.ConclusionsYPR2 is an important regulator balancing secondary metabolism with carbon metabolism in darkness and depending on the carbon source. The function of YPR2 reaches beyond the SOR cluster in which ypr2 is located and happens downstream of carbon catabolite repression mediated by CRE1.

Spontaneous phenotypic suppression of GacA-defective Vibrio fischeri is achieved via mutation of csrA and ihfA.

BackgroundSymbiosis defective GacA-mutant derivatives of Vibrio fischeri are growth impaired thereby creating a selective advantage for growth-enhanced spontaneous suppressors. Suppressors were isolated and characterized for effects of the mutations on gacA-mutant defects of growth, siderophore activity and luminescence. The mutations were identified by targeted and whole genome sequencing.ResultsMost mutations that restored multiple phenotypes were non-null mutations that mapped to conserved domains in or altered expression of CsrA, a post-transcriptional regulator that mediates GacA effects in a number of bacterial species. These represent an array of unique mutations compared to those that have been described previously. Different substitutions at the same amino acid residue were identified allowing comparisons of effects such as at the R6 residue, which conferred relative differences in luminescence and siderophore levels. The screen revealed residues not previously identified as critical for function including a single native alanine. Most csrA mutations enhanced luminescence more than siderophore activity, which was especially evident for mutations predicted to reduce the amount of CsrA. Although CsrA mutations compensate for many known GacA mutant defects, not all CsrA suppressors restore symbiotic colonization. Phenotypes of a suppressor allele of ihfA that encodes one subunit of the integration host factor (IHF) heteroduplex indicated the protein represses siderophore and activates luminescence in a GacA-independent manner.ConclusionsIn addition to its established role in regulation of central metabolism, the CsrA regulator represses luminescence and siderophore as an intermediate of the GacA regulatory hierachy. Siderophore regulation was less sensitive to stoichiometry of CsrA consistent with higher affinity for the targets of this trait. The lack of CsrA null-mutant recovery implied these mutations do not enhance fitness of gacA mutants and alluded to this gene being conditionally essential. This study also suggests a role for IHF in the GacA-CsrB-CsrA regulatory cascade by potentially assisting with the binding of repressors of siderohphore and activators of luminescence. As many phosphorelay proteins reduce fitness when mutated, the documented instability used in this screen also highlights a potentially universal and underappreciated problem that, if not identified and strategically avoided, could introduce confounding variability during experimental study of these regulatory pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0509-2) contains supplementary material, which is available to authorized users.

Transcriptomic profiling-based mutant screen reveals three new transcription factors mediating menadione resistance in Neurospora crassa

To gain insight into the regulatory mechanisms of oxidative stress responses in filamentous fungi, the genome-wide transcriptional response of Neurospora crassa to menadione was analysed by digital gene expression (DGE) profiling, which identified 779 upregulated genes and 576 downregulated genes. Knockout mutants affecting 130 highly-upregulated genes were tested for menadione sensitivity, which revealed that loss of the transcription factor siderophore regulation (SRE) (a transcriptional repressor for siderophore biosynthesis), catatase-3, cytochrome c peroxidase or superoxide dismutase 1 copper chaperone causes hypersensitivity to menadione. Deletion of sre dramatically increased transcription of the siderophore biosynthesis gene ono and the siderophore iron transporter gene sit during menadione stress, suggesting that SRE is required for repression of iron uptake under oxidative stress conditions. Contrary to its phenotype, the sre deletion mutant showed higher transcriptional levels of genes encoding reactive oxygen species (ROS) scavengers than wild type during menadione stress, which implies that the mutant suffers a higher level of oxidative stress than wild type. Uncontrolled iron uptake in the sre mutant might exacerbate cellular oxidative stress. This is the first report of a negative regulator of iron assimilation participating in the fungal oxidative stress response. In addition to SRE, eight other transcription factor genes were also menadione-responsive but their single gene knockout mutants showed wild-type menadione sensitivity. Two of them, named as mit-2 (menadione induced transcription factor-2) and mit-4 (menadione induced transcription factor-4), were selected for double mutant analysis. The double mutant was hypersensitive to menadione. Similarly, the double mutation of mit-2 and sre also had additive effects on menadione sensitivity, suggesting multiple transcription factors mediate oxidative stress resistance in an additive manner.

Combat of iron-deprivation through a plant growth promoting fluorescent Pseudomonas strain GRP3A in mung bean ( Vigna radiata L. Wilzeck)

Microorganisms and plants sustain themselves under iron-deprived conditions by releasing siderophores. Among others, fluorescent pseudomonads are known to exert extensive biocontrol action against soil and root borne phytopathogens through release of antimicrobials and siderophores. In this study, production and regulation of siderophores by fluorescent Pseudomonas strain GRP3A was studied. Among various media tested, standard succinate medium (SSM) promoted maximum siderophore production of 56.59 mg l(-1). There were low levels of siderophore in complex media like King's B medium, trypticase soya medium and nutrient medium (41.27, 29.86 and 27.63 mg l(-1)), respectively. In defferrated SSM, siderophore level was quantified to be 68.74 mg l(-1). Supplementation with iron (FeCl3) resulted in decreased siderophore levels depending on concentration. Siderophore production was promoted by Zn2+ (78.94 mg l(-1)), Cu2+ (68.80 mg l(-1)) whereas Co2+ (57.33 mg l(-1)) and Fe3+ reduced siderophore production (37.44 mg l(-1) as compared to control (55.97 mg l(-1)). Strain GRP3A showed plant growth promotion under iron limited conditions.

Role of the ferric uptake regulator of Pseudomonas aeruginosa in the regulation of siderophores and exotoxin A expression: purification and activity on iron-regulated promoters.

The cloned Pseudomonas aeruginosa fur (ferric uptake regulator) gene was overexpressed in P. aeruginosa by using a T7 expression system, and the Fur protein (PA-Fur) was purified by using a combination of ion-exchange chromatography and metal affinity chromatography. The DNA binding activity of the PA-Fur protein was confirmed by gel mobility shift assays and DNase I footprints of the synthetic DNA fragment GATAAT GATAATCATTATC, representing a perfect "Fur box". In addition, it was shown that PA-Fur is capable of binding to promoter and operator determinants of the tightly iron-regulated Escherichia coli fepA-fes enterobactin gene system. The activity of PA-Fur on the promoters of iron-regulated genes involved in the production of two siderophores, pyochelin and pyoverdin, and in the expression of exotoxin A was investigated. Data indicating that the promoters of the pchR gene, encoding a transcriptional activator for pyochelin synthesis, and of the pvdS gene, encoding a positive regulator for pyoverdin production, are specifically recognized by Fur-Fe(II) are presented, suggesting that PA-Fur represses expression of pchR and pvdS during growth in an iron-replete environment. However, neither the promoter region of the gene encoding exotoxin A (toxA) nor the promoters of the regAB operon, required for toxA expression, interacted with high concentrations of purified PA-Fur. These data indicate that iron regulation of exotoxin A production involves additional factors which may ultimately be under the control of PA-Fur.

Alginate regulatory and biosynthetic gene homologs in pseudomonas putida WCS358: correlation with the siderophore regulatory gene pfrA

A previous study [Venturi et al., Mol. Microbiol. 10 (1993) 63–73] demonstrated that the siderophore regulatory gene pfrA of Pseudomonas putida (Pp) WCS358 is highly similar and interchangeable with the alginate regulatory gene algQ ( algR2) of P. aeruginosa (Pa). The a1gQ gene is physically linked to two other alginate regulators in the Pa chromosome, namely algR ( algR1) , a response regulator, and algP ( algR3), a histone-like gene. In this study, we have identified the same genes and a similar genetic organization in the Pp chromosome. The two genes linked to pf rA, designated pprA and pprB, are similar to algR and algP, respectively. Chromosomal mutants of pprA and pprB were constructed showing that unlike pf rA, the two newly identified regulators are not involved in siderophore regulation. The pprA gene complemented a Pa algR mutant phenotype, suggesting that it could be involved in alginate gene regulation. The WCS358 strain is not producing alginate, but we demonstrated by Southern analysis that it also possesses, in addition to pprA and pprB, algD and algU ( algT) gene homologs, two genes essential for alginate biosynthesis. Using an algD-xylE transcriptional fusion, we observed that the algD promoter is active in strain WCS358 and absolutely requires pfrA. The possibility that all five genes of Pp WCS358 are involved in alginate biosynthesis is discussed.