Iron-depleted Conditions Research Articles

Staphylococcal sRNA IsrR downregulates methylthiotransferase MiaB under iron-deficient conditions.

Staphylococcus aureus is a major contributor to bacterial-associated mortality, owing to its exceptional adaptability across diverse environments. Iron is vital to most organisms but can be toxic in excess. To manage its intracellular iron, S. aureus, like many pathogens, employs intricate systems. We have recently identified IsrR as a key regulatory RNA induced during iron starvation. Its role is to reduce the synthesis of non-essential iron-containing proteins under iron-depleted conditions. In this study, we unveil IsrR's regulatory action on MiaB, an enzyme responsible for methylthio group addition to specific sites on transfer RNAs (tRNAs). We use predictive tools and reporter fusion assays to demonstrate IsrR's binding to the Shine-Dalgarno sequence of miaB RNA, thereby impeding its translation. The effectiveness of IsrR hinges on the integrity of a specific C-rich region. As MiaB is non-essential and has iron-sulfur clusters, IsrR induction spares iron by downregulating miaB. This may improve S. aureus fitness and aid in navigating the host's nutritional immune defenses.IMPORTANCEIn many biotopes, including those found within an infected host, bacteria confront the challenge of iron deficiency. They employ various strategies to adapt to this scarcity of nutrients, one of which involves regulating iron-containing proteins through the action of small regulatory RNAs. Our study shows how IsrR, a small RNA from S. aureus, prevents the production of MiaB, a tRNA-modifying enzyme containing iron-sulfur clusters. With this illustration, we propose a new substrate for an iron-sparing small RNA, which, when downregulated, should reduce the need for iron and save it to essential functions.

Escherichia coli monothiol glutaredoxin GrxD replenishes Fe-S clusters to the essential ErpA A-type carrier under low iron stress

Iron-sulfur (Fe-S) clusters are required for essential biological pathways, including respiration and isoprenoid biosynthesis. Complex Fe-S cluster biogenesis systems have evolved to maintain an adequate supply of this critical protein cofactor. In Escherichia coli, two Fe-S biosynthetic systems, the “housekeeping” Isc and “stress responsive” Suf pathways, interface with a network of cluster trafficking proteins, such as ErpA, IscA, SufA, and NfuA. GrxD, a Fe-S cluster-binding monothiol glutaredoxin, also participates in Fe-S protein biogenesis in both prokaryotes and eukaryotes. Previous studies in E. coli showed that the ΔgrxD mutation causes sensitivity to iron depletion, spotlighting a critical role for GrxD under conditions that disrupt Fe-S homeostasis. Here, we utilized a global chemoproteomic mass spectrometry (MS) approach to analyse the contribution of GrxD to the Fe-S proteome. Our results demonstrate that 1) GrxD is required for biogenesis of a specific subset of Fe-S proteins under iron-depleted conditions, 2) GrxD is required for cluster delivery to ErpA under iron limitation, 3) GrxD is functionally distinct from other Fe-S trafficking proteins and, 4) GrxD Fe-S cluster binding is responsive to iron limitation. All these results lead to the proposal that GrxD is required to maintain Fe-S cluster delivery to the essential trafficking protein ErpA during iron limitation conditions.

Two ferrous iron transporter-like proteins independently participate in asexual development under iron limitation and virulence in Beauveria bassiana

Reductive assimilation pathway involves ferric reductase and ferrous iron transporter, which is integral for fungal iron acquisition. A family of ferric reductase-like proteins has been functionally characterized in the filamentous entomopathogenic fungus Beauveria bassiana. In this investigation, two ferrous iron transporter-like proteins (Ftr) were functionally annotated in B. bassiana. BbFtr1 and BbFtr2 displayed high similarity in structure and were associated with the plasma and nuclear membrane. Their losses had no negatively influence on fungal growth on various nutrients and development under the iron-replete condition. Single mutants of BbFTR1 and BbFTR2 displayed the iron-availability dependent developmental defects, and double mutant exhibited the significantly impaired developmental potential under the iron-limited conditions. In insect bioassay, the double mutant also showed the weaker virulence than either of two single disruption mutants. These results suggested that two ferrous iron transporter-like proteins function independently in fungal physiologies under the iron-deficient condition. Intriguingly, a bZIP transcription factor BbHapX was required for expression of BbFTR1 and BbFTR2 under iron-depleted conditions. This study enhances our understanding of the iron uptake system in the filamentous entomopathogenic fungi.

HemU and TonB1 contribute to hemin acquisition in Stenotrophomonas maltophilia.

The hemin acquisition system is composed of an outer membrane TonB-dependent transporter that internalizes hemin into the periplasm, periplasmic hemin-binding proteins to shuttle hemin, an inner membrane transporter that transports hemin into the cytoplasm, and cytoplasmic heme oxygenase to release iron. Fur and HemP are two known regulators involved in the regulation of hemin acquisition. The hemin acquisition system of Stenotrophomonas maltophilia is poorly understood, with the exception of HemA as a TonB-dependent transporter for hemin uptake. Putative candidates responsible for hemin acquisition were selected via a homolog search and a whole-genome survey of S. maltophilia. Operon verification was performed by reverse transcription-polymerase chain reaction. The involvement of candidate genes in hemin acquisition was assessed using an in-frame deletion mutant construct and iron utilization assays. The transcript levels of candidate genes were determined using quantitative polymerase chain reaction. Smlt3896-hemU-exbB2-exbD2-tonB2 and tonB1-exbB1-exbD1a-exbD1b operons were selected as candidates for hemin acquisition. Compared with the parental strain, hemU and tonB1 mutants displayed a defect in their ability to use hemin as the sole iron source for growth. However, hemin utilization by the Smlt3896 and tonB2 mutants was comparable to that of the parental strain. HemA expression was repressed by Fur in iron-replete conditions and derepressed in iron-depleted conditions. HemP negatively regulated hemA expression. Like hemA, hemU was repressed by Fur in iron-replete conditions; however, hemU was moderately derepressed in response to iron-depleted stress and fully derepressed when hemin was present. Unlike hemA and hemU, the TonB1-exbB1-exbD1a-exbD1b operon was constitutively expressed, regardless of the iron level or the presence of hemin, and Fur and HemP had no influence on its expression. HemA, HemU, and TonB1 contribute to hemin acquisition in S. maltophilia. Fur represses the expression of hemA and hemU in iron-replete conditions. HemA expression is regulated by low iron levels, and HemP acts as a negative regulator of this regulatory circuit. HemU expression is regulated by low iron and hemin levels in a hemP-dependent manner.

NCOA4 requires a [3Fe-4S] to sense and maintain the iron homeostasis

NCOA4 is a selective cargo receptor for ferritinophagy, the autophagic turnover of ferritin (FTH), a process critical for regulating intracellular iron bioavailability. However, how ferritinophagy flux is controlled through NCOA4 in iron-dependent processes needs to be better understood. Here, we show that the C-terminal FTH-binding domain of NCOA4 harbors a [3Fe-4S] binding site with a stoichiometry of approximately one labile [3Fe-4S] cluster per NCOA4 monomer. By analyzing the interaction between NCOA4 and HERC2 ubiquitin ligase or NCOA4 and FTH, we demonstrate that NCOA4 regulates ferritinophagy by sensing the intracellular iron-sulfur-cluster levels. Under iron repletion conditions, HERC2 recognizes and recruits holo-NCOA4 as a substrate for polyubiquitination and degradation, favoring ferritin iron storage. Under iron depletion conditions, NCOA4 exists in the form of apo-protein and binds ferritin to promote the occurrence of ferritinophagy and release iron. Thus, we identify an iron-sulfur cluster [3Fe-4S] as a critical cofactor in determining the fate of NCOA4 in favoring iron storage in ferritin or iron release via ferritinophagy and provide a dual mechanism for selective interaction between HERC2 and [3Fe-4S]-NCOA4 for proteasomal degradation or between ferritin and apo-NCOA4 for ferritinophagy in the control of iron homeostasis.

High efficacy and enhanced synergistic activity of the novel siderophore-cephalosporin cefiderocol against multidrug-resistant and extensively drug-resistant Klebsiella pneumoniae from inpatients attending a single hospital in the United Arab Emirates

BackgroundCefiderocol (CFDC) is a novel siderophore-cephalosporin, which usually penetrates the bacteria through the iron-uptake pathways. Data is limited on the factors affecting CFDC activity and methods for overcoming resistance development. Synergistic approaches are needed to tackle antimicrobial resistance. This study aimed to determine CFDC activity on Klebsiella pneumoniae isolates from patients attending a single hospital in the United Arab Emirates (UAE), to explore the effect of β-lactamases on CFDC activity and to enhance CFDC susceptibility in both iron-depleted and iron-enriched conditions. MethodsWe investigated 238 K. pneumoniae strains from diverse clinical sources. β-lactamase genes were detected by PCR. Susceptibility to CFDC and 12 comparator antibiotics were tested. Combinations of CFDC with β-lactamase inhibitors (BLIs) and/or an outer membrane (OM) permeabilizer (polymyxin B nonapeptide) were tested in iron-depleted and iron-enriched conditions. ResultsCFDC exhibited efficacy of 97.9%, against multidrug-resistant (MDR), and extensively drug-resistant (XDR) strains, in addition to strains resistant to the last resort drugs such as colistin and tigecycline, including dual carbapenemase-producers (blaNDM and blaOXA-48-like) with MIC ≤ 0.06–8 µg/ml. It was effective in killing strains with single and multiple β-lactamases; however, it lost activity in iron-enriched conditions. Synergy was achieved with dual combination of CFDC and BLIs, especially avibactam, which caused a significant reduction in MICs even in iron-enriched conditions. A significant reduction was seen with the triple combination including an OM permeabilizer plus avibactam. Killing-kinetic studies proved that the combination therapy caused dose reduction and faster killing by CFDC than the monotherapy. ConclusionsCFDC was deemed effective against MDR and XDR K. pneumoniae. Synergistic combination of CFDC with BLIs and OM permeabilizers could be effective to treat infections in iron-rich sites, but this should be investigated in vivo.

Comparative metabolomic profiling of Cupriavidus necator B-4383 revealed production of cupriachelin siderophores, one with activity against Cryptococcus neoformans.

Cupriavidus necator H16 is known to be a rich source of linear lipopeptide siderophores when grown under iron-depleted conditions; prior literature termed these compounds cupriachelins. These small molecules bear β-hydroxyaspartate moieties that contribute to a photoreduction of iron when bound as ferric cupriachelin. Here, we present structural assignment of cupriachelins from C. necator B-4383 grown under iron limitation. The characterization of B-4383 cupriachelins is based on MS/MS fragmentation analysis, which was confirmed by 1D- and 2D-NMR for the most abundant analog (1). The cupriachelin congeners distinguish these two strains with differences in the preferred lipid tail; however, our rigorous metabolomic investigation also revealed minor analogs with changes in the peptide core, hinting at a potential mechanism by which these siderophores may reduce biologically unavailable ferric iron (4-6). Antifungal screening of the C. necator B-4383 supernatant extract and the isolated cupriachelin analog (1) revealed inhibitory activity against Cryptococcus neoformans, with IC50 values of 16.6 and 3.2μg/mL, respectively. This antifungal activity could be explained by the critical role of the iron acquisition pathway in the growth and pathogenesis of the C. neoformans fungal pathogen.

Exploring Antibiotic-Potentiating Effects of Tobramycin-Deferiprone Conjugates in Pseudomonas aeruginosa.

Metal ions, including Fe3+, affect the target site binding of some antibiotics and control the porin- and siderophore-mediated uptake of antibiotics. Amphiphilic tobramycins are an emerging class of antibiotic potentiators capable of synergizing with multiple classes of antibiotics against Gram-negative bacteria, including Pseudomonas aeruginosa. To study how the antibiotic-potentiating effect of amphiphilic tobramycins is affected by the presence of intermolecular iron chelators, we conjugated the FDA-approved iron chelator deferiprone (DEF) to tobramycin (TOB). Three TOB-DEF conjugates differing in the length of the carbon tether were prepared and tested for antibacterial activity and synergistic relationships with a panel of antibiotics against clinical isolates of P. aeruginosa. While all TOB-DEF conjugates were inactive against P. aeruginosa, the TOB-DEF conjugates strongly synergized with outer-membrane-impermeable antibiotics, such as novobiocin and rifampicin. Among the three TOB-DEF conjugates, 1c containing a C12 tether showed a remarkable and selective potentiating effect to improve the susceptibility of multidrug-resistant P. aeruginosa isolates to tetracyclines when compared with other antibiotics. However, the antibacterial activity and antibiotic-potentiating effect of the optimized conjugate was not enhanced under iron-depleted conditions, indicating that the function of the antibiotic potentiator is not affected by the Fe3+ concentration.

Rapid development of cefiderocol resistance in a carbapenem-resistant Pseudomonas aeruginosa isolate associated with mutations in the pyoverdine biosynthesis pathway

Here we report the in vivo development of cefiderocol resistance within 11 days after therapy initiation in a critically ill patient with bloodstream infection, infection of peri-anal fistula, and pneumonia caused by a VIM-2 harbouring, carbapenem-resistant Pseudomonas aeruginosa. Compared to a cefiderocol-naïve P. aeruginosa blood culture isolate, agar diffusion susceptibility testing found a reduced cefiderocol inhibition zone diameter in a P. aeruginosa recovered from peri-anal abscess tissue cultures after initiation of cefiderocol therapy. Subsequent whole-genome sequencing suggested that both isolates were of clonal origin. Comparison of genomes found an accumulation of missense mutations within pvdP, pvdE, pvdJ, and pvdD (i.e. genes associated with biosynthesis of pyoverdine), the main siderophore produced by P. aeruginosa. Quantification of pyoverdine production under iron-depleted conditions showed a significantly (P=0.0003) higher pyoverdine production by the cefiderocol-resistant isolate. While pyoverdine quantity alone appears not to be decisive for cefiderocol resistance, the reported case highlights the potentially rapid emergence of cefiderocol resistance in P. aeruginosa and points towards a potential involvement of iron up-take systems in this process.

Monitoring Fe-S cluster occupancy across the E. coli proteome using chemoproteomics.

Iron-sulfur (Fe-S) clusters are ubiquitous metallocofactors involved in redox chemistry, radical generation and gene regulation. Common methods to monitor Fe-S clusters include spectroscopic analysis of purified proteins and autoradiographic visualization of radiolabeled iron distribution in proteomes. Here, we report a chemoproteomic strategy that monitors changes in the reactivity of Fe-S cysteine ligands to inform on Fe-S cluster occupancy. We highlight the utility of this platform in Escherichia coli by (1) demonstrating global disruptions in Fe-S incorporation in cells cultured under iron-depleted conditions, (2) determining Fe-S client proteins reliant on five scaffold, carrier and chaperone proteins within the Isc Fe-S biogenesis pathway and (3) identifying two previously unannotated Fe-S proteins, TrhP and DppD. In summary, the chemoproteomic strategy described herein is a powerful tool that reports on Fe-S cluster incorporation directly within a native proteome, enabling the interrogation of Fe-S biogenesis pathways and the identification of previously uncharacterized Fe-S proteins.

Highly Metastatic Subpopulation of TNBC Cells Has Limited Iron Metabolism and Is a Target of Iron Chelators.

Excess iron is known to be a risk factor of carcinogenesis. Although iron chelators show anti-cancer effects, they have not been used successfully to treat cancer patients. Triple-negative breast cancer (TNBC) is a disease with poor prognosis without effective treatments. Thus, we aimed to evaluate a possibility of iron chelators as a therapy for TNBC. Deferasirox (DFX), an iron chelator, suppressed the growth of 4T1 murine TNBC cell line cells in vitro and in vivo. Lung metastasis was further significantly reduced, leading to the hypothesis that iron metabolism between metastatic and non-metastatic cells may be different. An analysis of existing database demonstrated that the expression of iron-uptake genes was significantly suppressed in TNBC cells that metastasized to lymph nodes or lungs compared to those in primary tumors. A highly metastatic clone of the murine 4T1 TNBC cells (4T1-HM) did not proliferate well under iron-rich or iron-depleted conditions by iron chelators compared to a low-metastatic clone (4T1-LM). Bulk RNA-seq analysis of RNA from 4T1-HM and 4T1-LM cells suggested that the PI3K-AKT pathway might be responsible for this difference. Indeed, DFX suppressed the proliferation via the AKT-mTOR pathway in 4T1-HM and the human MDA-MB-231 cells, a human mesenchymal-like TNBC cell line. DFX also suppressed the growth of 4T1-HM tumors in comparison to 4T1-LM tumors, and reduced lung metastases after surgical resection of primary 4T1 tumors. These results indicated, for the first time, that highly metastatic TNBC cells have limited iron metabolism, and they can be more effectively targeted by iron chelators.

A Simple Semi-hydroponic System for Studying Iron Homeostasis in Maize.

Hydroponic-based systems for plant growth allow control of the nutrients that plants take up through the provided nutrient solution. Different formulations of nutrient solutions enable the study of nutrient deficiencies in plants. Here we describe a procedure for setting up a simple semi-hydroponic system to grow maize seedlings. The system can be set up on a small or large scale, depending on the number of individuals studied. A modified nutrient solution is used for growing maize seedlings in iron-replete and iron-depleted conditions. This setup allows for studies of iron-deficiency responses in maize.

Elucidating the effect of iron acquisition systems in Klebsiella pneumoniae on susceptibility to the novel siderophore-cephalosporin cefiderocol.

Cefiderocol (CFDC) is a novel siderophore-cephalosporin, effective against multidrug-resistant Gram-negative bacteria. As it has a siderophore side chain, it can utilize iron acquisition systems for penetration of the bacterial outer membrane. We aimed to elucidate the role of siderophores and iron uptake receptors in defining Klebsiella pneumoniae susceptibility to CFDC. Initially, 103 K. pneumoniae strains were characterized for susceptibility to different antibiotics including CFDC. CFDC minimum inhibitory concentrations (MIC) were determined in iron-depleted and iron-enriched conditions. Iron uptake genes including siderophores, their receptors, ferric citrate (fecA) and iron uptake (kfu) receptors were detected by PCR in all the strains. For 10 selected strains, gene expression was tested in iron-depleted media with or without CFDC treatment and compared to expression in iron-enriched conditions. CFDC exhibited 96.1% susceptibility, being superior to all the other antibiotics (MIC50: 0.5 and MIC90: 4 μg/ml). Only three strains (2.9%) were intermediately susceptible and a pandrug resistant strain (0.97%) was resistant to CFDC (MIC: 8 and 256 μg/ml, respectively). The presence of kfu and fecA had a significant impact on CFDC MIC, especially when co-produced, and if coupled with yersiniabactin receptor (fyuA). CFDC MICs were negatively correlated with enterobactin receptor (fepA) expression and positively correlated with expression of kfu and fecA. Thus, fepA was associated with increased susceptibility to CFDC, while kfu and fecA were associated with reduced susceptibility to CFDC. CFDC MICs increased significantly in iron-enriched media, with reduced expression of siderophore receptors, hence, causing less drug uptake. Iron acquisition systems have a significant impact on CFDC activity, and their altered expression is a factor leading to reduced susceptibility. Iron concentration is also a major player affecting CFDC susceptibility; therefore, it is essential to explore possible ways to improve the drug activity to facilitate its use to treat infections in iron-rich sites.

Functional Portrait of Irf1 (Orf19.217), a Regulator of Morphogenesis and Iron Homeostasis in Candida albicans.

The alternate growth of Candida albicans between a unicellular yeast form and a multicellular hyphal form is crucial for its ability to cause disease. Interestingly, both morphological forms support distinct functions during proliferation in the human host. We previously identified ORF19.217 (C2_08890W_A), encoding a zinc-finger transcription factor of the C2H2 family, in a systematic screen of genes whose overexpression contributes to C. albicans’ morphological changes. Conditional overexpression of ORF19.217 with the strong tetracycline-inducible promoter (P TET ) resulted in a hyperfilamentous phenotype. We examined growth of the orf19.217 knockout-mutant in different hypha-inducing conditions and found that the mutant still formed hyphae under standard hypha-inducing conditions. To further investigate the function of Orf19.217 in C. albicans, we combined genome-wide expression (RNA-Seq) and location (ChIP-Seq) analyses. We found that Orf19.217 is involved in regulatory processes comprising hyphal morphogenesis and iron acquisition. Comparative analysis with existing C. albicans hyphal transcriptomes indicates that Orf19.217-mediated filamentation is distinct from a true hyphal program. Further, the orf19.217 knockout-mutant did not show increased sensitivity to iron deprivation, but ORF19.217 overexpression was able to rescue the growth of a hap5-mutant, defective in a subunit of the CCAAT-complex, which is essential for iron acquisition. This suggested that Orf19.217 is involved in regulation of iron acquisition genes during iron deprivation and acts in a parallel pathway to the established CCAAT-complex. Interestingly, the orf19.217-mutant turned out to be defective in its ability to form filaments under iron-deficiency. Taken together our findings propose that the transcription factor Orf19.217 stimulates expression of the hyphal regulators EFG1 and BRG1 to promote filamentous growth under iron deprivation conditions, allowing the fungus to escape these iron-depleted conditions. The transcription factor therefore appears to be particularly important for adaptation of C. albicans to diverse environmental conditions in the human host. In regard to the newly identified functions, we have given the regulator the name Irf1, Iron-dependent Regulator of Filamentation.

Iron deficiency aggravates DMNQ-induced cytotoxicity via redox cycling in kidney-derived cells

Iron, an essential element for most of living organisms, participates in many biological functions. Since iron is redox-active transition metal, it is known that excessive levels stimulate the formation of reactive oxygen species (ROS) and exacerbate cytotoxicity. An iron deficiency is the most common nutritional deficiency disorder in the world (about 30% of the population) and is more common than cases of iron overload. However, the effects of iron deficiency on ROS-induced cytotoxicity and the maintenance of intracellular redox homeostasis are not fully understood. The present study reports on an evaluation of the effects of iron deficiency on cytotoxicity induced by several ROS generators. In contrast to hydrogen peroxide and erastin, the cytotoxicity of 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a redox cycling agent that induces intracellular superoxide anion formation, was exacerbated by iron deficiency. Cytochrome b5 reductase was identified as a candidate enzyme responsible for the redox cycling of DMNQ under conditions of iron depletion. Moreover, the DMNQ-induced intracellular accumulation of ROS and a decrease in NADH/NAD+ ratios were enhanced by an iron deficiency. These negative changes were found to be ameliorated by overexpressing NAD(P)H:quinone oxidoreductase 1 (NQO1) in kidney-derived cells that originally showed a very low expression of NQO1. These results indicate that NQO1 plays a protective role against redox cycling quinone-mediated cytotoxicity under iron-depleted conditions. This is because NQO1 generates less-toxic hydroquinones via the two-electron reduction of quinones. The collective findings reported herein demonstrate that not only an iron overload but also an iron deficiency exacerbates ROS-mediated cytotoxicity.

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.

Identification and analysis of iron transporters from the fission yeast Schizosaccharomyces pombe.

Iron is an essential trace metal ion required for all living organisms, and is taken up by iron transporters. Here, we identified and characterized three-candidate high-affinity (Fio1, Frp1 and Frp2) and two-candidate low-affinity iron transporters (Fet4 and Pdt1) from the fission yeast Schizosaccharomyces pombe. Protein sequence analyses revealed that Fio1 is a multicopper oxidase that contains three cupredoxin domains with eleven candidate iron-binding ligands, whereas Frp1 harbors a ferric reductase domain with three-candidate heme-binding ligands. Protein sequence analyses also revealed that Fet4 and Pdt1 are integral membrane proteins with 10 and 11 transmembrane regions, respectively. Deletion of fio1 and, to a lesser extent, frp1 impaired growth under iron-depleted conditions, whereas deletion of frp1 and, to a lesser extent, frp2 inhibited growth under iron-replete conditions. Deletion of fet4 and pdt1 did not affect the growth of cells under iron-depleted and iron-replete conditions. Deletion of fio1 or frp1 also increased the sensitivity of cells to other transition metals. The copper sensitivity of Δfio1 cells could be rescued by iron, suggesting that the addition of iron mightdecrease the uptake of potentially toxic copper in Δfio1 cells. The copper sensitivity of Δfio1 cells could also be rescued by deletion of frp1, suggesting that Fio1 and Frp1 may function together in iron and copper uptakes in S. pombe. Our results revealed that iron and copper uptake systems may be partially overlapped in S. pombe.

The viability of Leptospira is related to physicochemical properties of the surface water surrounding an agricultural area and HemO and LipL32 gene expression in response to iron in water

The pathogenic Leptospira can survive and contaminate surface water based on physicochemical factors. This study aimed to determine how the physicochemical properties of water sources influence the growth and effect of iron on the gene expression of Leptospira spp. P47. Surface water samples (n=55) were collected and used for Leptospira spp. P47 cultivation. Physicochemical factors, including iron, calcium, magnesium and pH, were analyzed. The association between Leptospira spp. P47 viability at days 5, 10 and 15 with the physicochemical factors were analyzed. In addition, this bacterium was cultured in six selected water samples. The effect of iron in water on HemO and LipL32 gene expression was determined by relative quantification real-time PCR. Leptospira viability at day 5 was not significantly correlated with physicochemical factors, while Leptospira viability at day 10 was associated with both pH and iron. The Leptospira viability rate at day 15 had a significantly positive association with pH and iron and a negative association with calcium. HemO expression was significantly increased, mostly in selected water samples and under iron-depleted conditions. Conversely, LipL32 expression was significantly decreased in all water samples. Physicochemical factors in natural surface waters are key factors for bacterial survival in the environment, which may increase the chance of Leptospira infection in humans.

Maturation of UTR-Derived sRNAs Is Modulated during Adaptation to Different Growth Conditions.

Small regulatory RNAs play a major role in bacterial gene regulation by binding their target mRNAs, which mostly influences the stability or translation of the target. Expression levels of sRNAs are often regulated by their own promoters, but recent reports have highlighted the presence and importance of sRNAs that are derived from mRNA 3′ untranslated regions (UTRs). In this study, we investigated the maturation of 5′ and 3′ UTR-derived sRNAs on a global scale in the facultative phototrophic alphaproteobacterium Rhodobacter sphaeroides. Including some already known UTR-derived sRNAs like UpsM or CcsR1-4, 14 sRNAs are predicted to be located in 5 UTRs and 16 in 3′ UTRs. The involvement of different ribonucleases during maturation was predicted by a differential RNA 5′/3′ end analysis based on RNA next generation sequencing (NGS) data from the respective deletion strains. The results were validated in vivo and underline the importance of polynucleotide phosphorylase (PNPase) and ribonuclease E (RNase E) during processing and maturation. The abundances of some UTR-derived sRNAs changed when cultures were exposed to external stress conditions, such as oxidative stress and also during different growth phases. Promoter fusions revealed that this effect cannot be solely attributed to an altered transcription rate. Moreover, the RNase E dependent cleavage of several UTR-derived sRNAs varied significantly during the early stationary phase and under iron depletion conditions. We conclude that an alteration of ribonucleolytic processing influences the levels of UTR-derived sRNAs, and may thus indirectly affect their mRNA targets.