Iron piracy: acquisition of transferrin-bound iron by bacterial pathogens

The mechanism of iron utilization from transferrin has been most extensively characterized in the pathogenic Neisseria species and Haemophilus species. Two transferrin-binding proteins, Tbp1 and Tbp2, have been identified in these pathogens and are thought to be components of the transferrin receptor. Tbp1 appears to be an integral, TonB-dependent outer membrane protein while Tbp2, a lipoprotein, may be peripherally associated with the outer membrane. The relative contribution of each of these proteins to transferrin binding and utilization is discussed and a model of iron uptake from transferrin is presented. Sequence comparisons of the genes encoding neisserial transferrin-binding proteins suggest that they are probably under positive selection for variation and may have resulted from inter-species genetic exchange.     

Phenazine-1-carboxylic acid promotes bacterial biofilm development via ferrous iron acquisition

The opportunistic pathogen Pseudomonas aeruginosa forms biofilms, which render it more resistant to antimicrobial agents. Levels of iron in excess of what is required for planktonic growth have been shown to promote biofilm formation, and therapies that interfere with ferric iron [Fe(III)] uptake combined with antibiotics may help treat P. aeruginosa infections. However, use of these therapies presumes that iron is in the Fe(III) state in the context of infection. Here we report the ability of phenazine-1-carboxylic acid (PCA), a common phenazine made by all phenazine-producing pseudomonads, to help P. aeruginosa alleviate Fe(III) limitation by reducing Fe(III) to ferrous iron [Fe(II)]. In the presence of PCA, a P. aeruginosa mutant lacking the ability to produce the siderophores pyoverdine and pyochelin can still develop into a biofilm. As has been previously reported (P. K. Singh, M. R. Parsek, E. P. Greenberg, and M. J. Welsh, Nature 417:552-555, 2002), biofilm formation by the wild type is blocked by subinhibitory concentrations of the Fe(III)-binding innate-immunity protein conalbumin, but here we show that this blockage can be rescued by PCA. FeoB, an Fe(II) uptake protein, is required for PCA to enable this rescue. Unlike PCA, the phenazine pyocyanin (PYO) can facilitate biofilm formation via an iron-independent pathway. While siderophore-mediated Fe(III) uptake is undoubtedly important at early stages of infection, these results suggest that at later stages of infection, PCA present in infected tissues may shift the redox equilibrium between Fe(III) and Fe(II), thereby making iron more bioavailable.     

Rapid evolution of a polyphenic threshold

Polyphenisms are thought to play an important role in the evolution of phenotypic diversity and the origin of morphological and behavioral novelties. However, the extent to which polyphenic developmental mechanisms evolve in natural populations is unknown. Here we contrast patterns of male phenotype expression in native and exotic and ancestral and descendant populations of the horn polyphenic beetle, Onthophagus taurus. Males in this species express two alternative morphologies in response to larval feeding conditions. Favorable conditions cause males to grow larger than a threshold body size and to develop a pair of horns on their heads. Males that encounter relatively poor conditions do not reach this threshold size and remain hornless. We show that exotic and native populations of O. taurus differ significantly in the body size threshold that separates alternative male phenotypes. Comparison with archival museum collections and additional samples obtained from the native range of O. taurus suggests that allometric differences between exotic and native populations do not reflect preexisting variation in the native range of this species. Instead, our data suggest that threshold divergences between exotic and native populations have evolved in less than 40 years since the introduction to a new habitat and have proceeded in opposite directions in two exotic ranges of this species. Finally, we show that the kind and magnitude of threshold divergence between native and exotic populations are similar to differences normally observed between species. Our results support the view that certain components of the developmental control mechanism that underlie polyphenic development can evolve rapidly in natural populations and may provide important avenues for phenotypic differentiation and diversification in nature. We discuss the role of developmental control mechanisms in the origin of allometric diversification and explore potential evolutionary mechanisms that could drive scaling relationship evolution in nature.     

Regulation of iron acquisition and iron distribution in mammals

Both cellular iron deficiency and excess have adverse consequences. To maintain iron homeostasis, complex mechanisms have evolved to regulate cellular and extracellular iron concentrations. Extracellular iron concentrations are controlled by a peptide hormone hepcidin, which inhibits the supply of iron into plasma. Hepcidin acts by binding to and inducing the degradation of the cellular iron exporter, ferroportin, found in sites of major iron flows: duodenal enterocytes involved in iron absorption, macrophages that recycle iron from senescent erythrocytes, and hepatocytes that store iron. Hepcidin synthesis is in turn controlled by iron concentrations, hypoxia, anemia and inflammatory cytokines. The molecular mechanisms that regulate hepcidin production are only beginning to be understood, but its dysregulation is involved in the pathogenesis of a spectrum of iron disorders. Deficiency of hepcidin is the unifying cause of hereditary hemochromatoses, and excessive cytokine-stimulated hepcidin production causes hypoferremia and contributes to anemia of inflammation.     

Mycobactin-mediated iron acquisition within macrophages

Restricting the availability of iron is an important strategy for defense against bacterial infection. Mycobacterium tuberculosis survives within the phagosomes of macrophages; consequently, iron acquisition is particularly difficult for M. tuberculosis, because the phagosomal membrane is an additional barrier for its iron access. However, little is known about the iron transport and acquisition pathways adapted by this microbe in vivo. Extracellular iron sources are usually mobilized by hydrophilic siderophores. Here, we describe direct evidence that mycobactins, the lipophilic siderophores of mycobacteria, efficiently extract intracellular macrophage iron. The metal-free siderophore is diffusely associated with the macrophage membrane, ready for iron chelation. Notably, the mycobactin-metal complex accumulates with high selectivity in macrophage lipid droplets, intracellular domains for lipid storage and sorting. In our experiments, these mycobactin-targeted lipid droplets were found in direct contact with phagosomes, poised for iron delivery. The existence of this previously undescribed iron acquisition pathway indicates that mycobacteria have taken advantage of endogenous macrophage mechanisms for iron mobilization and lipid sorting for iron acquisition during infection. The pathway could represent a new target for the control of mycobacterial infection.     

PROFICS: A bacterial selection system for directed evolution of proteases

Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial selection system, which enables the optimization of proteases for biotechnology, therapeutics or diagnosis in a simple overnight process. During the PROFICS process, proteases are selected for their ability to specifically cut a tag from a reporter enzyme and leave a native N-terminus. Precise and efficient cleavage after the recognition sequence reverses the phenotype of an Escherichia coli knockout strain deficient in an essential enzyme of pyrimidine synthesis. A toolbox was generated to select for proteases with different preferences for P1′ residues (the residue immediately following the cleavage site). The functionality of PROFICS is demonstrated with viral proteases and human caspase-2. PROFICS improved caspase-2 activity up to 25-fold after only one round of mutation and selection. Additionally, we found a significantly improved tolerance for all P1′ residues caused by a mutation in a substrate interaction site. We showed that this improved activity enables cells containing the new variant to outgrow cells containing all other mutants, facilitating its straightforward selection. Apart from optimizing enzymatic activity and P1′ tolerance, PROFICS can be used to reprogram specificities, erase off-target activity, optimize expression via tags/codon usage, or even to screen for potential drug-resistance-conferring mutations in therapeutic targets such as viral proteases in an unbiased manner.     

Rapid molecular evolution in a living fossil

The tuatara of New Zealand is a unique reptile that coexisted with dinosaurs and has changed little morphologically from its Cretaceous relatives. Tuatara have very slow metabolic and growth rates, long generation times and slow rates of reproduction. This suggests that the species is likely to exhibit a very slow rate of molecular evolution. Our analysis of ancient and modern tuatara DNA shows that, surprisingly, tuatara have the highest rate of molecular change recorded in vertebrates. Our work also suggests that rates of neutral molecular and phenotypic evolution are decoupled.     

Rapid and onsite BOD sensing system using luminous bacterial cells-immobilized chip

A BOD monitoring system based on a bio-chip which immobilized luminous bacterium in micrometer-order holes were arrayed and fabricated by micro-machine techniques, was developed. The acrylic chip (3 cmx3 cm) comprises nine micro-holes (diameter: 700 microm or 1 mm, depth: 100 microm) arranged in a three by three array. Cells of the marine luminous bacterium, Photobacterium phosphoreum IFO 13896, which was grown at 15 degrees C for 15 h, were immobilized with 3% or 15% sodium alginate gel. BOD standard solutions or actual sample solution (approximately 10 microl) was fallen onto the cell-arrayed chip, and then the chip was incubated at 25 degrees C for 25 min. After incubation, bioluminescence from the each hole was gray-scaled and measured by a chemi-imager or newly developed onsite-type-monitoring system using a digital camera and a mobile-type personal computer. BOD values less than 16 ppm could detect by the chip, in particular, linear relationship at the concentrations between 0 and 16 ppm could be observed when luminous cells were immobilized with 3% sodium alginate gel. Steady bioluminescence was observed on the chip in the presence of BOD standard solution (GGA solution) which contained mineral elements. Furthermore, simultaneous detection of BOD values in various samples could be employed in the single chip. These results showed that the monitoring system with bio-chip could achieve high-through-put and onsite BOD detection. Our newly developed onsite-type BOD detection system which was used a digital camera and a (mobile) laptop computer was applied to measure and detect organic pollution due to biodegradable substances in wastewater treatment system. The same performance as the chemi-imager system was obtained for data of bioluminescence. The obtained BOD values showed a similar correlation with that of the conventional method for BOD determination (BOD5). These results suggested for successful achievement of high-though-put and onsite detection of BOD in practical.     

Reduction of iron by extracellular iron reductases: implications for microbial iron acquisition

The extracellular enzymatic reduction of iron by microorganisms has not been appropriately considered. In this study the reduction and release of iron from ferrioxamine were examined using extracellular microbial iron reductases and compared to iron mobilization by chemical reductants, and to chelation by EDTA and desferrioxamine. A flavin semiquinone was formed during the enzymatic reduction of ferrioxamine, which was consistent with the 1 e(-) reduction of iron by an enzyme. The rates for the enzymatic reactions were substantially faster than both the 2 e(-) chemical reductions and the chelation reactions. The rapid rates of the enzymatic reduction reactions demonstrated that these enzymes are capable of accomplishing the extracellular mobilization of iron required by microorganisms. The data suggest that mechanistically there are two phases for the mobilization and transport of iron by those microorganisms that produce both extracellular iron reductases and siderophores, with reduction being the principle pathway.     

Parallelising a model of bacterial interaction and evolution

Large simulations of bacterial colonies require huge amounts of computational time, the only way to achieve the necessary level of performance is with parallel computers and a suitably designed implementation that maps the problem onto the hardware. For real problems this mapping can be a non-trivial problem requiring careful consideration of the constraints in both the system being modelled and the hardware that executes that model. Here we describe an implementation of a system for modelling bacterial evolution that encompasses many physical scales. This system is composed entirely of individual entities all playing out a complex series of interactions. These individuals exist at the scale of the population of bacterial and at the gene product scale. This paper reports that it is possible to map a dynamic problem such as this onto fixed resources, for the most part making use of implicit multiplexing of resources provided by the OS and partitioning the problem to reduce communication time. Through this an efficient simulation can be created, making maximal use of the available hardware without constraining the model to require excessively specific resources.     

The mineral weathering ability of Collimonas pratensis PMB3(1) involves a Malleobactin-mediated iron acquisition system

Mineral weathering by microorganisms is considered to occur through a succession of mechanisms based on acidification and chelation. While the role of acidification is established, the role of siderophores is difficult to disentangle from the effect of the acidification. We took advantage of the ability of strain Collimonas pratensis PMB3(1) to weather minerals but not to acidify depending on the carbon source to address the role of siderophores in mineral weathering. We identified a single non-ribosomal peptide synthetase (NRPS) responsible for siderophore biosynthesis in the PMB3(1) genome. By combining iron-chelating assays, targeted mutagenesis and chemical analyses (HPLC and LC-ESI-HRMS), we identified the siderophore produced as malleobactin X and how its production depends on the concentration of available iron. Comparison with the genome sequences of other collimonads evidenced that malleobactin production seems to be a relatively conserved functional trait, though some collimonads harboured other siderophore synthesis systems. We also revealed by comparing the wild-type strain and its mutant impaired in the production of malleobactin that the ability to produce this siderophore is essential to allow the dissolution of hematite under non-acidifying conditions. This study represents the first characterization of the siderophore produced by collimonads and its role in mineral weathering.     

革兰氏阴性菌亚铁离子转运系统的组成及作用机制

几乎所有细菌的生长都离不开铁元素。在有氧的环境中,三价铁离子几乎无法被细菌直接利用。但是在宿主胃肠道中,铁元素主要以可溶性的亚铁离子形式存在,它们可通过革兰氏阴性菌外膜直接进入胞周质,在周质通过亚铁离子转运系统,将铁离子转运至胞浆供细菌利用。绝大多数阴性菌主要是通过Feo转运系统利用亚铁离子,大肠杆菌的Feo转运系统由feoA、feoB和feoC3个基因组成。除Feo转运系统外,还发现Yfe转运系统、Efe转运系统、Sit转运系统等。本文重点介绍革兰氏阴性菌Feo转运系统的组成及作用机制,以期为进一步研究细菌亚铁离子的转运机制提供参考。     

Transferrin-mediated iron acquisition by pathogenic Neisseria

The pathogenic Neisseria have a siderophore-independent iron-uptake system reliant on a direct interaction between the bacterial cell and transferrin. In the meningococcus this uptake system is dependent on two surface-exposed transferrin-binding proteins. This short account will review our current knowledge of the transferrin-mediated iron-acquisition system of pathogenic Neisseria.     

Strategies of plants for acquisition of iron

Two different types of root response to Fe deficiency (strategies) have been identified in species of the Plant Kingdom. In Strategy I which occurs in all plant species except grasses, a plasma membrane-bound reductase is induced with enhanced net excretion of protons. Often the release of reductants/chelators is also higher. In Strategy II which is confined to grasses, there is an increase in the biosynthesis and secretion of phytosiderophores which form chelates with Fe^III. Uptake of Fe^III phytosiderophores is mediated by a specific transporter in the plasma membrane of root cells of grasses. From results based mainly on long-term studies under non-axenic conditions this classification into two strategies has been questioned, and the utilization of Fe from microbial siderophores has been considered as an alternative strategy particularly in grasses. Possible reasons for controversial results are discussed in some detail. The numerous effects of microorganisms in non-axenic cultures, and the as yet inadequate characterization of the so-called standard (basic) reductase present major limitations to understanding different mechanisms of Fe acquisition. In comparison with the progress made in identifying the cellular mechanisms of root responses in Strategy I and Strategy II plants, our understanding is poor concerning the processes taking place in the apoplasm of root rhizodermal cells and of the role of low-molecular-weight root exudates and siderophores in Fe acquisition of plants growing in soils of differing Fe availability.