Functional expression of Escherichia coli fhuA gene in Rhizobium spp. of Cajanus cajan provides growth advantage in presence of Fe3+: ferrichrome as iron source

Cajanus cajan rhizobial isolates were found to be unable to utilize iron bound to ferrichrome, desferrioxamine B or rhodotorulic acid, all being hydroxamate type siderophores. A broad host range expression vector containing the Escherichia coli fhuA gene, encoding the outer membrane receptor for Fe-ferrichrome, was constructed. The plasmid construct (pGR1), designed to express fhuA under the lac promoter of E. coli, complemented E. coli MB97 ΔfhuA mutant for ferri-ferrichrome utilization and also allowed Rhizobium spp. ST1 and Rhizobium spp. IC3123 to grow using iron bound to ferrichrome. Sensitivity to the antibiotic albomycin, transported via the FhuA receptor, was found in case of MB97 as well as rhizobial transformants harboring pGR1. The rhizobial transformants expressing fhuA showed growth stimulation when co-inoculated with Ustilago maydis, a fungal species known to produce ferrichrome under iron starved conditions. Growth stimulation was also observed in the presence of externally supplied ferrichrome. The significance of these findings in terms of the potential for improving the survivability of rhizobial bioinoculant strains in natural soils is discussed.     

Hydroxamate Production as a High Affinity Iron Acquisition Mechanism in Paracoccidioides Spp

Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity.     

Utilization of siderophores by Candida albicans

Candida albicans secretes both hydroxamate and phenolate-type siderophores when grown under iron-restricted conditions. The inhibition of candidal growth by iron limitation was reversed by the addition of supplemental hydroxamate on phenolate siderophores. Both siderophores produced equal stimulation of growth suggesting that C. albicans could utilize both siderophores with equal efficiency. Addition of heterologous siderophores from both bacteria and fungi also supported growth of the yeast in a deferrated medium. These results suggest that C. albicans has an iron-uptake mechanism which enables it to obtain iron by utilizing candidal and non-candidal siderophores.     

Multiple roles of siderophores in free-living nitrogen-fixing bacteria

Free-living nitrogen-fixing bacteria in soils need to tightly regulate their uptake of metals in order to acquire essential metals (such as the nitrogenase metal cofactors Fe, Mo and V) while excluding toxic ones (such as W). They need to do this in a soil environment where metal speciation, and thus metal bioavailability, is dependent on a variety of factors such as organic matter content, mineralogical composition, and pH. Azotobacter vinelandii, a ubiquitous gram-negative soil diazotroph, excretes in its external medium catechol compounds, previously identified as siderophores, that bind a variety of metals in addition to iron. At low concentrations, complexes of essential metals (Fe, Mo, V) with siderophores are taken up by the bacteria through specialized transport systems. The specificity and regulation of these transport systems are such that siderophore binding of excess Mo, V or W effectively detoxifies these metals at high concentrations. In the topsoil (leaf litter layer), where metals are primarily bound to plant-derived organic matter, siderophores extract essential metals from natural ligands and deliver them to the bacteria. This process appears to be a key component of a mutualistic relationship between trees and soil diazotrophs, where tree-produced leaf litter provides a living environment rich in organic matter and micronutrients for nitrogen-fixing bacteria, which in turn supply new nitrogen to the ecosystem.     

Microbial pathogens with impaired ability to acquire host iron

Successful microbial pathogens must be adept in obtaining growth-essential iron from healthy hosts. Some potential pathogens, however, are sufficiently impaired in iron acquisition ability so as to be dangerous mainly in hosts with such iron loading conditions as alcoholism, asplenia, hemochromatosis, -thalassemia major, or tobacco smoking. The association of six impaired pathogens (Capnocytophaga canimorsis, Yersinia enterocolitica and Y. pseudotuberculosis, Vibrio vulnificus, Tropheryma whippelii, and Legionella pneumophila) with iron loaded humans is described.     

Siderophore production and utilization byRhizobium trifolii

Summary Several strains ofRhizobium trifolii were tested for their ability to synthesize and utilize phenolate or hydroxamate types of siderophores. None of the nodulating strains ofR. trifolii was able to produce detectable amounts of siderophores. Only the non-nodulating strainR. trifolii AR6 formed a phenolate siderophore, which stimulated the growth of the siderophore-negative mutant AR65. Other strains ofR. trifolii could not utilize iron from exogenously supplied Desferal, pseudobactin or citrate. The siderophore fromR. trifolii AR6 and 2,3-dihydroxybenzoic acid slightly stimulated the growth of someR. trifolii strains.     

Root-based N2-fixing Symbioses: Legumes, Actinorhizal Plants, Parasponia sp. and Cycads

In the mutualistic symbioses between legumes and rhizobia, actinorhizal plants and Frankia, Parasponia sp. and rhizobia, and cycads and cyanobacteria, the N₂-fixing microsymbionts exist in specialized structures (nodules or cyanobacterial zones) within the roots of their host plants. Despite the phylogenetic diversity among both the hosts and the microsymbionts of these symbioses, certain developmental and physiological imperatives must be met for successful mutualisms. In this review, phylogenetic and ecological aspects of the four symbioses are first addressed, and then the symbioses are contrasted and compared in regard to infection and symbio-organ development, supply of carbon to the microsymbionts, regulation of O₂ flux to the microsymbionts, and transfer of fixed-N to the hosts. Although similarities exist in the genetics, development, and functioning of the symbioses, it is evident that there is great diversity in many aspects of these root-based N₂-fixing symbioses. Each symbiosis can be admired for the elegant means by which the host plant and microsymbiont integrate to form the mutualistic relationships so important to the functioning of the biosphere.     

Iron and siderophores in fungal–host interactions

Most fungi and bacteria express specific mechanisms for the acquisition of iron from the hosts they infect for their own survival. This is primarily because iron plays a key catalytic role in various vital cellular reactions in conjunction with the fact that iron is not freely available in these environments due to host sequestration. High-affinity iron uptake systems, such as siderophore-mediated iron uptake and reductive iron assimilation, enable fungi to acquire limited iron from animal or plant hosts. Regulating iron uptake is crucial to maintain iron homeostasis, a state necessary to avoid iron-induced toxicity from iron abundance, while simultaneously supplying iron required for biochemical demand. Siderophores play diverse roles in fungal–host interactions, many of which have been principally delineated from gene deletions in non-ribosomal peptide synthetases, enzymes required for siderophore biosynthesis. These analyses have demonstrated that siderophores are required for virulence, resistance to oxidative stress, asexual/sexual development, iron storage, and protection against iron-induced toxicity in some fungal organisms. In this review, the strategies fungi employ to obtain iron, siderophore biosynthesis, and the regulatory mechanisms governing iron homeostasis will be discussed with an emphasis on siderophore function and relevance for fungal organisms in their interactions with their hosts.     

Iron uptake in pseudorevertants of Escherichia coli K-12 mutants with multiple defects in the enterochelin system

Iron uptake in pseudorevertants of Escherichia coli K-12 strains which lack the ability to synthesize enterochelin, 2,3-dihydroxybenzoate, and the ferrienterochelin receptor protein was characterized. In four independent pseudorevertants, the suppressor mutations which permitted growth in iron-poor environments appeared to be located in ompB, the regulatory locus for the porin proteins. Unlike wild-type cells, the pseudorevertants were unable to utilize ferrienterochelin and could acquire iron from citrate without induction by prior growth in citrate. The energy requirements of the pseudorevertant system appeared to be identical to those of the enterochelin system. Evidence that loss of the porin proteins results in the secretion by the pseudorevertants of a molecule with siderophore activity is presented; this siderophore is able to remove iron from the non-biological iron chelators nitrilotriacetic acid and , -dipyridyl but not from the siderophores ferrichrome and enterochelin.     

Siderophore-mediated iron acquisition in the staphylococci

Iron is frequently a growth-limiting nutrient due to its propensity to interact with oxygen to form insoluble precipitates and, therefore, biological systems have evolved specialized uptake mechanisms to obtain this essential nutrient. Many pathogenic bacteria are capable of obtaining stringently sequestered iron from animal hosts by one or both of the following mechanisms: extraction of heme from host erythrocyte and serum hemoproteins, or through the use of high affinity, iron-scavenging molecules termed siderophores. This review summarizes our current knowledge of siderophore-mediated iron acquisition systems in the genus Staphylococcus.     

Iron and virulence in Shigella

Iron limitation, a condition encountered within mammalian hosts, induces the synthesis of a number of proteins in pathogenic Shigella species. These include several outer membrane proteins, Shiga toxin, and proteins involved in the biosynthesis and transport of high-affinity iron-binding compounds or siderophores. Although siderophores have been shown to play a major role in the virulence of some bacterial pathogens, these compounds do not appear to be essential for the virulence of Shigella species. Unlike those pathogens which are restricted to the extracellular compartments of the host, the Shigella species invade and multiply within host cells. Alternative iron-acquisition systems, such as the ability to utilize haem-iron, permit growth of the intracellular bacteria. Virulent shigellae also possess a cell-surface haem-binding protein, and synthesis of this protein correlates with infectivity and virulence. This protein does not appear to be involved in iron acquisition. Rather, it may allow the bacteria to coat themselves with haem compounds, thus enhancing their ability to interact with target host cells.     

Iron, infection, and neoplasia

In nearly all forms of life, the number and diversity of enzymes that contain iron or that depend on the presence of this metal for activity are impressive. Not surprisingly, chemical mechanisms have been evolved by many organisms that permit them to solubilize and acquire iron while at the same time depriving their competitors or their pathogens of this element. Proteins such as transferrin and lactoferrin that are employed by vertebrate hosts for iron transport and acquisition can, to some extent, withhold the metal from the siderophores of invading bacteria and fungi. Attempts also are made by animal hosts to withhold iron from protozoa and neoplastic cells. Unfortunately, pathogenic microorganisms have developed a variety of counter measures that are especially dangerous in hosts stressed by iron overload in specific fluids, tissues, or cells. In recent years, however, a number of possible methods and agents for strengthening iron-withholding defense have become apparent. Nearly 3,000 papers on various aspects of iron withholding are contained in the 18-year Medline Database and numerous reviews have been published since 1966. The present paper will focus on developments that have been reported within the past 2 1/2 years.     

Vertical transmission as the key to the colonization of Madagascar by fungus-growing termites?

The mutualism between fungus-growing termites (Macrotermitinae) and their mutualistic fungi (Termitomyces) began in Africa. The fungus-growing termites have secondarily colonized Madagascar and only a subset of the genera found in Africa is found on this isolated island. Successful long-distance colonization may have been severely constrained by the obligate interaction of the termites with fungal symbionts and the need to acquire these symbionts secondarily from the environment for most species (horizontal symbiont transmission). Consistent with this hypothesis, we show that all extant species of fungus-growing termites of Madagascar are the result of a single colonization event of termites belonging to one of the only two groups with vertical symbiont transmission, and we date this event at approximately 13 Mya (Middle/Upper Miocene). Vertical symbiont transmission may therefore have facilitated long-distance dispersal since both partners disperse together. In contrast to their termite hosts, the fungal symbionts have colonized Madagascar multiple times, suggesting that the presence of fungus-growing termites may have facilitated secondary colonizations of the symbiont. Our findings indicate that the absence of the right symbionts in a new environment can prevent long-distance dispersal of symbioses relying on horizontal symbiont acquisition.     

Charcoal and shrubs modify soil processes in ponderosa pine forests of western Montana

In split-root systems of alfalfa (Medicago sativa L.), already existing nodules or arbuscular mycorrhizal roots suppress further establishment of symbiosis in other root parts, a phenomenon named autoregulation. Roots treated with rhizobial nodulation signals (Nod factors) induce a similar systemic suppression of symbiosis.In order to test the hypothesis that flavonoids play a role in this systemic suppression, split-root systems of alfalfa plants were inoculated on one side of the split-root system with Sinorhizobium meliloti or Glomus mosseae or were treated with Nod factor. HPLC-analysis of alfalfa root extracts from both sides of the split-root system revealed a persistent local and systemic accumulation pattern of some flavonoids associated with the different treatments. The two flavonoids, formononetin and ononin, could be identified to be similarily altered after rhizobial or mycorrhizal inoculation or when treated with Nod factor.Exogenous application of formononetin and ononin partially restored nodulation and mycorrhization pointing towards the involvement of these two secondary compounds in the autoregulation of both symbioses.     

Second-generation sequencing for gene discovery in the Brassicaceae

The Brassicaceae contains the most diverse collection of agriculturally important crop species of all plant families. Yet, this is one of the few families that do not form functional symbiotic associations with mycorrhizal fungi in the soil for improved nutrient acquisition. The genes involved in this symbiosis were more recently recruited by legumes for symbiotic association with nitrogen-fixing rhizobia bacteria. This study applied second-generation sequencing (SGS) and analysis tools to discover that two such genes, NSP1 (Nodulation Signalling Pathway 1) and NSP2, remain conserved in diverse members of the Brassicaceae despite the absence of these symbioses. We demonstrate the utility of SGS data for the discovery of putative gene homologs and their analysis in complex polyploid crop genomes with little prior sequence information. Furthermore, we show how this data can be applied to enhance downstream reverse genetics analyses. We hypothesize that Brassica NSP genes may function in the root in other plant-microbe interaction pathways that were recruited for mycorrhizal and rhizobial symbioses during evolution.     

Ferritins, bacterial virulence and plant defence

The enterobacterial pathogen Erwinia chrysanthemi causes soft rot diseases on a wide range of plants, including the model plant Arabidopsis thaliana. This bacterium proliferates in the host by secreting a set of pectin degrading enzymes responsible for symptom development. In addition, survival of this bacterium in planta requires two high-affinity iron acquisition systems mediated by siderophores and protective systems against oxidative damages, suggesting the implication by both partners of accurate mechanisms controlling their iron homeostasis under conditions of infection. In this review, we address this question and we show that ferritins both from the pathogen and the host are subtly implicated in the control of this interplay.     

Improving crop nutrient efficiency through root architecture modifications

Improving crop nutrient ef ficiency becomes an essential consideration for environmentally friendly and sustainable agriculture. Plant growth and development is dependent on 17 essential nutrient elements,among them,nitrogen(N) and phosphorus(P) are the two most important mineral nutrients. Hence it is not surprising that low N and/or low P availability in soils severely constrains crop growth and productivity,and thereby have become high priority targets for improving nutrient ef ficiency in crops. Root exploration largely determines the ability of plants to acquire mineral nutrients from soils. Therefore,root architecture,the 3-dimensional con figuration of the plant's root system in the soil,is of great importance for improving crop nutrient ef ficiency. Furthermore,the symbiotic associations between host plants and arbuscular mycorrhiza fungi/rhizobial bacteria,are additional important strategies to enhance nutrient acquisition. In this review,we summarize the recent advances in the current understanding of crop species control of root architecture alterations in response to nutrient availability and root/microbe symbioses,through gene or QTL regulation,which results in enhanced nutrient acquisition.     

Efficiency of partner choice and sanctions in Lotus is not altered by nitrogen fertilization

Eukaryotic hosts must exhibit control mechanisms to select against ineffective bacterial symbionts. Hosts can minimize infection by less-effective symbionts (partner choice) and can divest of uncooperative bacteria after infection (sanctions). Yet, such host-control traits are predicted to be context dependent, especially if they are costly for hosts to express or maintain. Legumes form symbiosis with rhizobia that vary in symbiotic effectiveness (nitrogen fixation) and can enforce partner choice as well as sanctions. In nature, legumes acquire fixed nitrogen from both rhizobia and soils, and nitrogen deposition is rapidly enriching soils globally. If soil nitrogen is abundant, we predict host control to be downregulated, potentially allowing invasion of ineffective symbionts. We experimentally manipulated soil nitrogen to examine context dependence in host control. We co-inoculated Lotus strigosus from nitrogen depauperate soils with pairs of Bradyrhizobium strains that vary in symbiotic effectiveness and fertilized plants with either zero nitrogen or growth maximizing nitrogen. We found efficient partner choice and sanctions regardless of nitrogen fertilization, symbiotic partner combination or growth season. Strikingly, host control was efficient even when L. strigosus gained no significant benefit from rhizobial infection, suggesting that these traits are resilient to short-term changes in extrinsic nitrogen, whether natural or anthropogenic.     

Multiple Symbiont Acquisition Strategies as an Adaptive Mechanism in the Coral Stylophora pistillata

In obligate symbioses, the host’s survival relies on the successful acquisition and maintenance of symbionts. Symbionts can either be transferred from parent to offspring via direct inheritance (vertical transmission) or acquired anew each generation from the environment (horizontal transmission). With vertical symbiont transmission, progeny benefit by not having to search for their obligate symbionts, and, with symbiont inheritance, a mechanism exists for perpetuating advantageous symbionts. But, if the progeny encounter an environment that differs from that of their parent, they may be disadvantaged if the inherited symbionts prove suboptimal. Conversely, while in horizontal symbiont acquisition host survival hinges on an unpredictable symbiont source, an individual host may acquire genetically diverse symbionts well suited to any given environment. In horizontal acquisition, however, a potentially advantageous symbiont will not be transmitted to subsequent generations. Adaptation in obligate symbioses may require mechanisms for both novel symbiont acquisition and symbiont inheritance. Using denaturing-gradient gel electrophoresis and real-time PCR, we identified the dinoflagellate symbionts (genus Symbiodinium) hosted by the Red Sea coral Stylophora pistillata throughout its ontogenesis and over depth. We present evidence that S. pistillata juvenile colonies may utilize both vertical and horizontal symbiont acquisition strategies. By releasing progeny with maternally derived symbionts, that are also capable of subsequent horizontal symbiont acquisition, coral colonies may acquire physiologically advantageous novel symbionts that are then perpetuated via vertical transmission to subsequent generations. With symbiont inheritance, natural selection can act upon the symbiotic variability, providing a mechanism for coral adaptation.     

Characterization of siderophore-mediated iron transport inGeotrichum candidum,a non-siderophore producer

Summary Geotrichum candidum is capable of utilizing iron from hydroxamate siderophores of different structural classes. The relative rates of iron transport for ferrichrome, ferrichrysin, ferrioxamine B, fusigen, ferrichrome A, rhodotorulic acid, coprogen B, dimerium acid and ferrirhodin were 100%, 98%, 74%, 59%, 49%, 35%, 24%, 12% and 11% respectively. Ferrichrome, ferrichrysine and ferrichrome A inhibited [⁵⁹Fe]ferrioxamine-B-mediated iron transport by 71%, 68% and 28% respectively when added at equimolar concentrations to the radioactive complex. The inhibitory mechanism of [⁵⁹Fe]ferrioxamine B uptake by ferrichrome was non-competitive (K _i 2.4 M), suggesting that the two siderophores do not share a common transport system. Uptake of [⁵⁹Fe]ferrichrome, [⁵⁹Fe]rhodotorulic acid and [⁵⁹Fe]fusigen was unaffected by competition with the other two siderophores or with ferrioxamine B. Thus,G. candidum may possess independent transport systems for siderophores of different structural classes. The uptake rates of [¹⁴C]ferrioxamine B and⁶⁷Ga-desferrioxamine B were 30% and 60% respectively, as compared to [⁵⁹Fe]ferrioxamine B. The specific ferrous chelates, dipyridyl and ferrozine at 6 mM, caused 65% and 35% inhibition of [⁵⁹Fe]ferrioxamine uptake. From these results we conclude that, although about 70% of the iron is apparently removed from the complex by reduction prior to being transported across the cellular membrane, a significant portion of the chelated ligand may enter the cell intact. The former and latter mechanisms seem not to be mutually exclusive.