Iron-limiting Conditions Research Articles

Iron restriction increases the expression of a cytotoxic cysteine proteinase TvCP2 by a novel mechanism of tvcp2 mRNA alternative polyadenylation in Trichomonas vaginalis

Trichomonas vaginalis TvCP2 (TVAG_057000) is a cytotoxic cysteine proteinase (CP) expressed under iron-limited conditions. This work aimed to identify one of the mechanisms of tvcp2 gene expression regulation by iron at the posttranscriptional level. We checked tvcp2 mRNA stability under both iron-restricted (IR) and high iron (HI) conditions in the presence of actinomycin D. Greater stability of the tvcp2 mRNA under the IR than in HI conditions was observed, as expected. In silico analysis of the 3′ regulatory region showed the presence of two putative polyadenylation signals in the tvcp2 transcript. By 3′-RACE assays, we demonstrated the existence of two isoforms of the tvcp2 mRNA with different 3′-UTR that resulted in more TvCP2 protein under IR than in HI conditions detected by WB assays. Additionally, we searched for homologs of the trichomonad polyadenylation machinery by an in silico analysis in the genome database, TrichDB. 16 genes that encode proteins that could be part of the trichomonad polyadenylation machinery were found. qRT-PCR assays showed that most of these genes were positively regulated by iron. Thus, our results show the presence of alternative polyadenylation as a novel iron posttranscriptional regulatory mechanism in T. vaginalis for the tvcp2 gene expression.

The Regulatory Hub of Siderophore Biosynthesis in the Phytopathogenic Fungus Alternaria alternata.

A GATA zinc finger-containing repressor (AaSreA) suppresses siderophore biosynthesis in the phytopathogenic fungus Alternaria alternata under iron-replete conditions. In this study, targeted gene deletion revealed two bZIP-containing transcription factors (AaHapX and AaAtf1) and three CCAAT-binding proteins (AaHapB, AaHapC, and AaHapE) that positively regulate gene expression in siderophore production. This is a novel phenotype regarding Atf1 and siderophore biosynthesis. Quantitative RT-PCR analyses revealed that only AaHapX and AaSreA were regulated by iron. AaSreA and AaHapX form a transcriptional feedback negative loop to regulate iron acquisition in response to the availability of environmental iron. Under iron-limited conditions, AaAtf1 enhanced the expression of AaNps6, thus playing a positive role in siderophore production. However, under nutrient-rich conditions, AaAtf1 plays a negative role in resistance to sugar-induced osmotic stress, and AaHapX plays a negative role in resistance to salt-induced osmotic stress. Virulence assays performed on detached citrus leaves revealed that AaHapX and AaAtf1 play no role in fungal pathogenicity. However, fungal strains carrying the AaHapB, AaHapC, or AaHapE deletion failed to incite necrotic lesions, likely due to severe growth deficiency. Our results revealed that siderophore biosynthesis and iron homeostasis are regulated by a well-organized network in A. alternata.

Fur functions as an activator of T6SS-mediated bacterial dominance and virulence in Aeromonas hydrophila.

Aeromonas hydrophila, a ubiquitous bacterium in aquatic habitats with broad host ranges, has earned the nickname of a 'Jack-of-all-trades'. However, there is still a limited understanding of the mechanism of how this bacterium fit the competition with other species in dynamic surroundings. The type VI secretion system (T6SS) is macromolecular machinery found in Gram-negative bacteria's cell envelope that is responsible for bacterial killing and/or pathogenicity toward different host cells. In this study, the depression of A. hydrophila T6SS under iron-limiting conditions was detected. The ferric uptake regulator (Fur) was then found to act as an activator of T6SS by directly binding to the Fur box region in vipA promoter in the T6SS gene cluster. The transcription of vipA was repressed in Δfur. Moreover, the inactivation of Fur resulted in considerable defects in the interbacterial competition activity and pathogenicity of A. hydrophila in vitro and in vivo. These findings provide the first direct evidence that Fur positively regulates the expression and functional activity of T6SS in Gram-negative bacteria and will help to understand the fascinating mechanism of competitive advantage for A. hydrophila in different ecological niches.

RyhB Paralogs Downregulate the Expressions of Multiple Survival-Associated Genes and Attenuate the Survival of Salmonella Enteritidis in the Chicken Macrophage HD11

RyhB-1 and RyhB-2 are small non-coding RNAs in Salmonella that act as regulators of iron homeostasis by sensing the environmental iron concentration. Expressions of RyhB paralogs from Salmonella Typhimurium are increased within microphages. RyhB paralogs restrain the growth of S. Typhimurium in RAW264.7 macrophages by modulating the expression of Salmonella pathogenicity island 1 (SPI-1) genes sicA and rtsB. However, little is known about the regulatory role of RyhBs and their virulence-associated targets in Salmonella Enteritidis. We studied candidate targets of RyhB paralogs via RNA-Seq in conditions of iron limitation and hypoxia. RyhB paralogs were expressed when the S. Enteritidis strain CMCC(B)50336 (SE50336) interacted with the chicken macrophage line HD11. We analyzed gene expression associated with Salmonella survival and replication in macrophages in wild-type strain SE50336 and the RyhB deletion mutants after co-incubation with HD11 and screened out targets regulated by RyhBs. The expressions of both RyhB-1 and RyhB-2 were increased after co-incubation with HD11 for 8 h and several survival-associated genes within macrophages, such as ssaI, sseA, pagC, sodC, mgtC, yaeB, pocR, and hns, were upregulated in the ryhB-1 deletion mutant. Specifically, ssaI, the type-three secretion system 2 (T3SS-2) effector encoded by SPI-2, which promoted the survival of Salmonella in macrophages, was upregulated more than 3-fold in the ryhB-1 deletion mutant. We confirmed that both RyhB-1 and RyhB-2 downregulated the expression of ssaI to repress its mRNA translation by directly interacting with its coding sequence (CDS) region via an incomplete complementary base-pairing mechanism. The SPI-2 gene sseA was indirectly modulated by RyhB-1. The survival assays in macrophages showed that the ability of intracellular survival of ryhB-1 and/or ryhB-2 deletion mutants in HD11 was higher than that of the wild-type strain. These results indicate that RyhB paralogs downregulate survival-related virulence factors and attenuate the survival of S. Enteritidis inside chicken macrophage HD11.

Role of peptidoglycan recycling enzymes AmpD and AnmK in Acinetobacter baumannii virulence features.

Acinetobacter baumannii is an important causative agent of hospital acquired infections. In addition to acquired resistance to many currently-available antibiotics, it is intrinsically resistant to fosfomycin. It has previously been shown that AmpD and AnmK contribute to intrinsic fosfomycin resistance in A. baumannii due to their involvement in the peptidoglycan recycling pathway. However, the role that these two enzymes play in the fitness and virulence of A. baumannii has not been studied. The aim of this study was to characterize several virulence-related phenotypic traits in A. baumannii mutants lacking AmpD and AnmK. Specifically, cell morphology, peptidoglycan thickness, membrane permeability, growth under iron-limiting conditions, fitness, resistance to disinfectants and antimicrobial agents, twitching motility and biofilm formation of the mutant strains A. baumannii ATCC 17978 ΔampD::Kan and ΔanmK::Kan were compared to the wild type strain. Our results demonstrate that bacterial growth and fitness of both mutants were compromised, especially in the ΔampD::Kan mutant. In addition, biofilm formation was decreased by up to 69%, whereas twitching movement was reduced by about 80% in both mutants. These results demonstrate that, in addition to increased susceptibility to fosfomycin, alteration of the peptidoglycan recycling pathway affects multiple aspects related to virulence. Inhibition of these enzymes could be explored as a strategy to develop novel treatments for A. baumannii in the future. Furthermore, this study establishes a link between intrinsic fosfomycin resistance mechanisms and bacterial fitness and virulence traits.

Diurnally dynamic iron allocation promotes N2 fixation in marine dominant diazotroph Trichodesmium

Trichodesmium is the dominant photoautotrophic dinitrogen (N2) fixer (diazotroph) in the ocean. Iron is an important factor limiting growth of marine diazotrophs including Trichodesmium mainly because of high iron content of its N2-fixing enzyme, nitrogenase. However, it still lacks a quantitative understanding of how dynamic iron allocation among physiological processes acts to regulate growth and N2 fixation in Trichodesmium. Here, we constructed a model of Trichodesmium trichome in which intracellular iron could be dynamically re-allocated in photosystems and nitrogenase during the daytime. The results demonstrate that the dynamic iron allocation enhances modeled N2 fixation and growth rates of Trichodesmium, especially in iron-limited conditions, albeit having a marginal impact under high iron concentrations. Although the reuse of iron during a day is an apparent cause that dynamic iron allocation can benefit Trichodesmium under iron limitation, our model reveals two important mechanisms. First, the release of iron from photosystems downregulates the intracellular oxygen (O2) production and reduces the demand of respiratory protection, a process that Trichodesmium wastefully respires carbohydrates to create a lower O2 window for N2 fixation. Hence, more carbohydrates can be used in growth. Second, lower allocation of iron to nitrogenase during early daytime, a period when photosynthesis is active and intracellular O2 is high, reduces the amount of iron that is trapped in the inactivated nitrogenase induced by O2. This mechanism further increases the iron use efficiency in Trichodesmium. Overall, our study provides mechanistic and quantitative insight into the diurnal iron allocation that can alleviate iron limitation to Trichodesmium.

In vivo characterisation of the Vibrio vulnificus stressosome: A complex involved in reshaping glucose metabolism and motility regulation, in nutrient- and iron-limited growth conditions.

Stressosomes are signal-sensing and integration hubs identified in many bacteria. At present, the role of the stressosome has only been investigated in Gram-positive bacteria. This work represents the first in vivo characterisation of the stressosome in a Gram-negative bacterium, Vibrio vulnificus. Previous in vitro characterisation of the complex has led to the hypothesis of a complex involved in iron metabolism and control of c-di-GMP levels. We demonstrate that the stressosome is probably involved in reshaping the glucose metabolism in Fe- and nutrient-limited conditions and mutations of the locus affect the activation of the glyoxylate shunt. Moreover, we show that the stressosome is needed for the transcription of fleQ and to promote motility, consistent with the hypothesis that the stressosome is involved in regulating c-di-GMP. This report highlights the potential role of the stressosome in a Gram-negative bacterium, with implications for the metabolism and motility of this pathogen.

Modification of 4-Fold and B-Pores in Bacterioferritin from Mycobacterium tuberculosis Reveals Their Role in Fe2+ Entry and Oxidoreductase Activity

The self-assembled ferritin nanocages, nature's solution to iron toxicity and its low solubility, scavenge free iron to synthesize hydrated ferric oxyhydroxide mineral inside their central cavity by protein-mediated ferroxidase and hydrolytic/nucleation reactions. These complex processes in ferritin commence with the rapid influx of Fe2+ ions via the inter-subunit contact points (i.e., pores/channels). Investigation of these pores as Fe2+ uptake routes in ferritins remains a subject of intense research, in iron metabolism, toxicity, and bacterial pathogenesis, which are yet to be established in the bacterioferritin (BfrA) from Mycobacterium tuberculosis (Mtb). The electrostatic properties of this protein indicate that the 4-fold and B-pores might serve as potential Fe2+ entry routes. Therefore, in the current work, electrostatics at/along these pores was altered by site-directed mutagenesis to establish their role in Fe2+ uptake/oxidation (ferroxidase activity) in Mtb BfrA. Despite forming self-assembled protein nanocompartment, these 4-fold and B-pore variants exhibited partial loss of ferroxidase activity and lower accumulation of transient species, which not only indicated their role in Fe2+ entry but also suggested the existence of multiple pathways. Although the B-pore variants inhibited the rapid ferroxidase activity to a larger extent, they had minimal impact on their cage stability. The current work revealed the relative contribution of these pores toward rapid Fe2+ uptake/oxidation and cage stability, possibly as consequences of their differential symmetry, number of modified residues (at each pore), and heme content. Therefore, these findings may help to understand the role of these pores in iron acquisition and Mtb proliferation under iron-limiting conditions to control its pathogenesis.

Flavobacterium columnare ferric iron uptake systems are required for virulence.

Flavobacterium columnare, which causes columnaris disease, is one of the costliest pathogens in the freshwater fish-farming industry. The virulence mechanisms of F. columnare are not well understood and current methods to control columnaris outbreaks are inadequate. Iron is an essential nutrient needed for metabolic processes and is often required for bacterial virulence. F. columnare produces siderophores that bind ferric iron for transport into the cell. The genes needed for siderophore production have been identified, but other components involved in F. columnare iron uptake have not been studied in detail. We identified the genes encoding the predicted secreted heme-binding protein HmuY, the outer membrane iron receptors FhuA, FhuE, and FecA, and components of an ATP binding cassette (ABC) transporter predicted to transport ferric iron across the cytoplasmic membrane. Deletion mutants were constructed and examined for growth defects under iron-limited conditions and for virulence against zebrafish and rainbow trout. Mutants with deletions in genes encoding outer membrane receptors, and ABC transporter components exhibited growth defects under iron-limited conditions. Mutants lacking multiple outer membrane receptors, the ABC transporter, or HmuY retained virulence against zebrafish and rainbow trout mirroring that exhibited by the wild type. Some mutants predicted to be deficient in multiple steps of iron uptake exhibited decreased virulence. Survivors of exposure to such mutants were partially protected against later infection by wild-type F. columnare.

Iron Deficiency Promotes the Lack of Photosynthetic Cytochrome c550 and Affects the Binding of the Luminal Extrinsic Subunits to Photosystem II in the Diatom Phaeodactylum tricornutum.

In the diatom Phaeodactylum tricornutum, iron limitation promotes a decrease in the content of photosystem II, as determined by measurements of oxygen-evolving activity, thermoluminescence, chlorophyll fluorescence analyses and protein quantification methods. Thermoluminescence experiments also indicate that iron limitation induces subtle changes in the energetics of the recombination reaction between reduced QB and the S2/S3 states of the water-splitting machinery. However, electron transfer from QA to QB, involving non-heme iron, seems not to be significantly inhibited. Moreover, iron deficiency promotes a severe decrease in the content of the extrinsic PsbV/cytochrome c550 subunit of photosystem II, which appears in eukaryotic algae from the red photosynthetic lineage (including diatoms) but is absent in green algae and plants. The decline in the content of cytochrome c550 under iron-limiting conditions is accompanied by a decrease in the binding of this protein to photosystem II, and also of the extrinsic PsbO subunit. We propose that the lack of cytochrome c550, induced by iron deficiency, specifically affects the binding of other extrinsic subunits of photosystem II, as previously described in cyanobacterial PsbV mutants.

Effect of sulfidation on nitrobenzene removal from groundwater by microscale zero-valent iron: Insights into reactivity, reaction sites and removal pathways

While it has been recognized that sulfidation can effectively improve the reactivity of microscale zero valent iron (mZVI), there is limited understanding of nitrobenzene (ArNO2) removal by sulfidated mZVI. To understand the reduction capacity and pathway of ArNO2 by sulfidated mZVI, ball-milling sulfidated mZVI (S-mZVIbm) with different S/Fe molar ratios (0–0.2) was used to conduct this experiment. The results showed that sulfidation could efficiently enhance ArNO2 removal under iron-limited and iron excess conditions, which was attributed to the presence of FeSx sites that could provide higher Fe(0) utilization efficiency and stronger passivation resisting for S-mZVIbm. The optimum ArNO2 reduction could be obtained by S-mZVIbm with S/Fe molar ratio at 0.1, which could completely transform ArNO2 to aniline (ArNH2) with a rate constant of 4.36 × 10−2 min−1 during 120-min reaction. FeSx phase could act as electron transfer sites for ArNO2 reduction and it could still be reserved in S-mZVIbm after reduction reaction. The product distribution indicated that sulfidation did not change the types of reduction products, while the removal of ArNO2 by S-mZVIbm was a step-by-step reduction progress along with the adsorption of ArNH2. In addition, a faster reduction of ArNO2 in groundwater/soil system further demonstrated the feasibility of S-mZVIbm in the real field remediation.

Iron Acquisition Mechanisms and Their Role in the Virulence of Acinetobacter baumannii.

Iron is an essential element for survival of most organisms. One mechanism of host defense is to tightly chelate iron to several proteins to limit its extracellular availability. This has forced pathogens such as Acinetobacter baumannii to adapt mechanisms for the acquisition and utilization of iron even in iron-limiting conditions. A. baumannii uses a variety of iron acquisition strategies to meet its iron requirements. It can lyse erythrocytes to harvest the heme molecules, use iron-chelating siderophores, and use outer membrane vesicles to acquire iron. Iron acquisition pathways, in general, have been seen to affect many other virulence factors such as cell adherence, cell motility, and biofilm formation. The knowledge gained from research on iron acquisition led to the synthesis of the antibiotic cefiderocol, which uses iron uptake pathways for entry into the cell with some success as a novel cephalosporin. Understanding the mechanisms of iron acquisition of A. baumannii allows for insight into clinical infections and offer potential targets for novel antibiotics or potentiators of current drugs.

Insights into Systems for Iron-Sulfur Cluster Biosynthesis in Acidophilic Microorganisms.

Fe-S clusters are versatile and essential cofactors that participate in multiple and fundamental biological processes. In Escherichia coli, the biogenesis of these cofactors requires either the housekeeping Isc pathway, or the stress-induced Suf pathway which plays a general role under conditions of oxidative stress or iron limitation. In the present work, the Fe-S cluster assembly Isc and Suf systems of acidophilic Bacteria and Archaea, which thrive in highly oxidative environments, were studied. This analysis revealed that acidophilic microorganisms have a complete set of genes encoding for a single system (either Suf or Isc). In acidophilic Proteobacteria and Nitrospirae, a complete set of isc genes (iscRSUAX-hscBA-fdx), but not genes coding for the Suf system, was detected. The activity of the Isc system was studied in Leptospirillum sp. CF-1 (Nitrospirae). RT-PCR experiments showed that eight candidate genes were co-transcribed and conform the isc operon in this strain. Additionally, RT-qPCR assays showed that the expression of the iscS gene was significantly up-regulated in cells exposed to oxidative stress imposed by 260 mM Fe2(SO4)3 for 1 h or iron starvation for 3 h. The activity of cysteine desulfurase (IscS) in CF-1 cell extracts was also up-regulated under such conditions. Thus, the Isc system from Leptospirillum sp. CF-1 seems to play an active role in stressful environments. These results contribute to a better understanding of the distribution and role of Fe-S cluster protein biogenesis systems in organisms that thrive in extreme environmental conditions.

ToxR is a c-di-GMP binding protein that modulates surface-associated behaviour in Pseudomonas aeruginosa

Pseudomonas aeruginosa uses multiple protein regulators that work in tandem to control the production of a wide range of virulence factors and facilitate rapid adaptation to diverse environmental conditions. In this opportunistic pathogen, ToxR was known to positively regulate the production of the major virulence factor exotoxin A and now, through analysis of genetic changes between two sublines of P. aeruginosa PAO1 and functional complementation of swarming, we have identified a previously unknown role of ToxR in surface-associated motility in P. aeruginosa. Further analysis revealed that ToxR had an impact on swarming motility by regulating the Rhl quorum sensing system and subsequent production of rhamnolipid surfactants. Additionally, ToxR was found to tightly bind cyclic diguanylate (c-di-GMP) and negatively affect traits controlled by this second messenger including reducing biofilm formation and the expression of Psl and Pel exopolysaccharides, necessary for attachment and sessile communities matrix scaffolding, in P. aeruginosa. Moreover, a link between the post-transcriptional regulator RsmA and toxR expression via the alternative sigma factor PvdS, induced under iron-limiting conditions, is established. This study reveals the importance of ToxR in a sophisticated regulation of free-living and biofilm-associated lifestyles, appropriate for establishing acute or chronic P. aeruginosa infections.

The NtrYX Two-Component System of Paracoccus denitrificans Is Required for the Maintenance of Cellular Iron Homeostasis and for a Complete Denitrification under Iron-Limited Conditions.

Denitrification consists of the sequential reduction of nitrate to nitrite, nitric oxide, nitrous oxide, and dinitrogen. Nitrous oxide escapes to the atmosphere, depending on copper availability and other environmental factors. Iron is also a key element because many proteins involved in denitrification contain iron-sulfur or heme centers. The NtrYX two-component regulatory system mediates the responses in a variety of metabolic processes, including denitrification. A quantitative proteomic analysis of a Paracoccus denitrificans NtrY mutant grown under denitrifying conditions revealed the induction of different TonB-dependent siderophore transporters and proteins related to iron homeostasis. This mutant showed lower intracellular iron content than the wild-type strain, and a reduced growth under denitrifying conditions in iron-limited media. Under iron-rich conditions, it releases higher concentrations of siderophores and displayes lower nitrous oxide reductase (NosZ) activity than the wild-type, thus leading to nitrous oxide emission. Bioinformatic and qRT-PCR analyses revealed that NtrYX is a global transcriptional regulatory system that responds to iron starvation and, in turn, controls expression of the iron-responsive regulators fur, rirA, and iscR, the denitrification regulators fnrP and narR, the nitric oxide-responsive regulator nnrS, and a wide set of genes, including the cd1-nitrite reductase NirS, nitrate/nitrite transporters and energy electron transport proteins.

Transcriptomic Response of the Diazotrophic Bacteria Gluconacetobacter diazotrophicus Strain PAL5 to Iron Limitation and Characterization of the fur Regulatory Network.

Gluconacetobacter diazotrophicus has been the focus of several studies aiming to understand the mechanisms behind this endophytic diazotrophic bacterium. The present study is the first global analysis of the early transcriptional response of exponentially growing G. diazotrophicus to iron, an essential cofactor for many enzymes involved in various metabolic pathways. RNA-seq, targeted gene mutagenesis and computational motif discovery tools were used to define the G. diazotrophicus fur regulon. The data analysis showed that genes encoding functions related to iron homeostasis were significantly upregulated in response to iron limitations. Certain genes involved in secondary metabolism were overexpressed under iron-limited conditions. In contrast, it was observed that the expression of genes involved in Fe-S cluster biosynthesis, flagellar biosynthesis and type IV secretion systems were downregulated in an iron-depleted culture medium. Our results support a model that controls transcription in G. diazotrophicus by fur function. The G. diazotrophicus fur protein was able to complement an E. coli fur mutant. These results provide new insights into the effects of iron on the metabolism of G. diazotrophicus, as well as demonstrate the essentiality of this micronutrient for the main characteristics of plant growth promotion by G. diazotrophicus.

Catecholate siderophore receptor CirA impacts cefiderocol susceptibility in Klebsiella pneumoniae

Cefiderocol is a cephalosporin antibiotic presenting expanded antimicrobial activity. CirA is a gateway for cefiderocol to enter bacterial cells. We found that cirA1 and cirA198 were primary alleles in Klebsiella pneumoniae. CirA1 exhibited higher iron-transporting ability than CirA198 in iron-limited conditions. The cefiderocol minimum inhibitory concentration (MIC) increased from 0.5 mg/L to 2 mg/L when cirA1 was mutated to cirA198. Consistently, the MIC showed a 4-fold decrease when cirA198 was mutated to cirA1. Therefore, CirA1 has higher capacity to transport siderophores, contributing to increased cefiderocol susceptibility.

Genome-wide characterization of Salmonella Typhimurium genes required for the fitness under iron restriction

BackgroundIron is a crucial element for bacterial survival and virulence. During Salmonella infection, the host utilizes a variety of mechanisms to starve the pathogen from iron. However, Salmonella activates distinctive defense mechanisms to acquire iron and survive in iron-restricted host environments. Yet, the comprehensive set of the conditionally essential genes that underpin Salmonella survival under iron-restricted niches has not been fully explored.ResultsHere, we employed transposon sequencing (Tn-seq) method for high-resolution elucidation of the genes in Salmonella Typhimurium (S. Typhimurium) 14028S strain required for the growth under the in vitro conditions with four different levels of iron restriction achieved by iron chelator 2,2′-dipyridyl (Dip): mild (100 and 150 μM), moderate (250 μM) and severe iron restriction (400 μM). We found that the fitness of the mutants reduced significantly for 28 genes, suggesting the importance of these genes for the growth under iron restriction. These genes include sufABCDSE, iron transport fepD, siderophore tonB, sigma factor E ropE, phosphate transport pstAB, and zinc exporter zntA. The siderophore gene tonB was required in mild and moderate iron-restricted conditions, but it became dispensable in severe iron-restricted conditions. Remarkably, rpoE was required in moderate and severe iron restrictions, leading to complete attenuation of the mutant under these conditions. We also identified 30 genes for which the deletion of the genes resulted in increased fitness under iron-restricted conditions.ConclusionsThe findings broaden our knowledge of how S. Typhimurium survives in iron-deficient environments, which could be utilized for the development of new therapeutic strategies targeting the pathways vital for iron metabolism, trafficking, and scavenging.

Siderophore for Lanthanide and Iron Uptake for Methylotrophy and Plant Growth Promotion in Methylobacterium aquaticum Strain 22A.

Methylobacterium and Methylorubrum species are facultative methylotrophic bacteria that are abundant in the plant phyllosphere. They have two methanol dehydrogenases, MxaF and XoxF, which are dependent on either calcium or lanthanides (Lns), respectively. Lns exist as insoluble minerals in nature, and their solubilization and uptake require a siderophore-like substance (lanthanophore). Methylobacterium species have also been identified as plant growth-promoting bacteria although the actual mechanism has not been well-investigated. This study aimed to reveal the roles of siderophore in Methylobacterium aquaticum strain 22A in Ln uptake, bacterial physiology, and plant growth promotion. The strain 22A genome contains an eight-gene cluster encoding the staphyloferrin B-like (sbn) siderophore. We demonstrate that the sbn siderophore gene cluster is necessary for growth under low iron conditions and was complemented by supplementation with citrate or spent medium of the wild type or other strains of the genera. The siderophore exhibited adaptive features, including tolerance to oxidative and nitrosative stress, biofilm formation, and heavy metal sequestration. The contribution of the siderophore to plant growth was shown by the repressive growth of duckweed treated with siderophore mutant under iron-limited conditions; however, the siderophore was dispensable for strain 22A to colonize the phyllosphere. Importantly, the siderophore mutant could not grow on methanol, but the siderophore could solubilize insoluble Ln oxide, suggesting its critical role in methylotrophy. We also identified TonB-dependent receptors (TBDRs) for the siderophore–iron complex, iron citrate, and Ln, among 12 TBDRs in strain 22A. Analysis of the siderophore synthesis gene clusters and TBDR genes in Methylobacterium genomes revealed the existence of diverse types of siderophores and TBDRs. Methylorubrum species have an exclusive TBDR for Ln uptake that has been identified as LutH. Collectively, the results of this study provide insight into the importance of the sbn siderophore in Ln chelation, bacterial physiology, and the diversity of siderophore and TBDRs in Methylobacterium species.

Plant-Derived Catechols Are Substrates of TonB-Dependent Transporters and Sensitize Pseudomonas aeruginosa to Siderophore-Drug Conjugates.

ABSTRACTPseudomonas aeruginosa is an opportunistic pathogen responsible for acute and chronic infections in immunocompromised hosts. This organism is known to compete efficiently against coinfecting microorganisms, due in part to the secretion of antimicrobial molecules and the synthesis of siderophore molecules with high affinity for iron. P. aeruginosa possess a large repertoire of TonB-dependent transporters for the uptake of its own, as well as xenosiderophores released from other bacteria or fungi. Here, we show that P. aeruginosa is also capable of utilizing plant-derived polyphenols as an iron source. We found that exclusively plant-derived phenols containing a catechol group (i.e., chlorogenic acid, caffeic acid, quercetin, luteolin) induce the expression of the TonB-dependent transporters PiuA or PirA. This induction requires the two-component system PirR-PirS. Chlorogenic acid in its Fe(III)-loaded form was actively transported by PiuA and PirA and supported growth under iron-limiting conditions. Coincidentally, PiuA and PirA are also the main TonB transporters for the recently approved siderophore-drug conjugate cefiderocol. Surprisingly, quercetin supplementation increased the susceptibility of P. aeruginosa to siderophore-drug conjugates, due to induction of piuA and pirA expression mediated by the PirR-PirS two-component system. These findings suggest a potential novel therapeutic application for these biologically active dietary polyphenols.