Suboxic Deposition of Ferric Iron by Bacteria in Opposing Gradients of Fe(II) and Oxygen at Circumneutral pH

The influence of lithotrophic Fe(II)-oxidizing bacteria on patterns of ferric oxide deposition in opposing gradients of Fe(II) and O₂ was examined at submillimeter resolution by use of an O₂ microelectrode and diffusion microprobes for iron. In cultures inoculated with lithotrophic Fe(II)-oxidizing bacteria, the majority of Fe(III) deposition occurred below the depth of O₂ penetration. In contrast, Fe(III) deposition in abiotic control cultures occurred entirely within the aerobic zone. The diffusion microprobes revealed the formation of soluble or colloidal Fe(III) compounds during biological Fe(II) oxidation. The presence of mobile Fe(III) in diffusion probes from live cultures was verified by washing the probes in anoxic water, which removed ca. 70% of the Fe(III) content of probes from live cultures but did not alter the Fe(III) content of probes from abiotic controls. Measurements of the amount of Fe(III) oxide deposited in the medium versus the probes indicated that ca. 90% of the Fe(III) deposited in live cultures was formed biologically. Our findings show that bacterial Fe(II) oxidation is likely to generate reactive Fe(III) compounds that can be immediately available for use as electron acceptors for anaerobic respiration and that biological Fe(II) oxidation may thereby promote rapid microscale Fe redox cycling at aerobic-anaerobic interfaces.     

Tomato TFT1 Is Required for PAMP-Triggered Immunity and Mutations that Prevent T3S Effector XopN from Binding to TFT1 Attenuate Xanthomonas Virulence

XopN is a type III effector protein from Xanthomonas campestris pathovar vesicatoria that suppresses PAMP-triggered immunity (PTI) in tomato. Previous work reported that XopN interacts with the tomato 14-3-3 isoform TFT1; however, TFT1''s role in PTI and/or XopN virulence was not determined. Here we show that TFT1 functions in PTI and is a XopN virulence target. Virus-induced gene silencing of TFT1 mRNA in tomato leaves resulted in increased growth of Xcv ΔxopN and Xcv ΔhrpF demonstrating that TFT1 is required to inhibit Xcv multiplication. TFT1 expression was required for Xcv-induced accumulation of PTI5, GRAS4, WRKY28, and LRR22 mRNAs, four PTI marker genes in tomato. Deletion analysis revealed that the XopN C-terminal domain (amino acids 344–733) is sufficient to bind TFT1. Removal of amino acids 605–733 disrupts XopN binding to TFT1 in plant extracts and inhibits XopN-dependent virulence in tomato, demonstrating that these residues are necessary for the XopN/TFT1 interaction. Phos-tag gel analysis and mass spectrometry showed that XopN is phosphorylated in plant extracts at serine 688 in a putative 14-3-3 recognition motif. Mutation of S688 reduced XopN''s phosphorylation state but was not sufficient to inhibit binding to TFT1 or reduce XopN virulence. Mutation of S688 and two leucines (L64,L65) in XopN, however, eliminated XopN binding to TFT1 in plant extracts and XopN virulence. L64 and L65 are required for XopN to bind TARK1, a tomato atypical receptor kinase required for PTI. This suggested that TFT1 binding to XopN''s C-terminal domain might be stabilized via TARK1/XopN interaction. Pull-down and BiFC analyses show that XopN promotes TARK1/TFT1 complex formation in vitro and in planta by functioning as a molecular scaffold. This is the first report showing that a type III effector targets a host 14-3-3 involved in PTI to promote bacterial pathogenesis.     

Evidence for independent recruitment of zeta-crystallin/quinone reductase (CRYZ) as a crystallin in camelids and hystricomorph rodents

Zeta-crystallin/quinone reductase (CRYZ) is an NADPH oxidoreductase expressed at very high levels in the lenses of two groups of mammals: camelids and some hystricomorph rodents. It is also expressed at very low levels in all other species tested. Comparative analysis of the mechanisms mediating the high expression of this enzyme/crystallin in the lens of the Ilama (Lama guanacoe) and the guinea pig (Cavia porcellus) provided evidence for independent recruitment of this enzyme as a lens crystallin in both species and allowed us to elucidate for the first time the mechanism of lens recruitment of an enzyme- crystallin. The data presented here show that in both species such recruitment most likely occurred through the generation of new lens promoters from nonfunctional intron sequences by the accumulation of point mutations and/or small deletions and insertions. These results further support the idea that recruitment of CRYZ resulted from an adaptive process in which the high expression of CRYZ in the lens provides some selective advantage rather than from a purely neutral evolutionary process.      

U(VI) Sequestration in Hydroxyapatite Produced by Microbial Glycerol 3-Phosphate Metabolism

Previous studies have demonstrated the potential for removal of U(VI) from solution via precipitation of U(VI)-bearing calcium-phosphate (Ca-P) minerals coupled to microbial hydrolysis of glycerol phosphate compounds. We evaluated this process in circumneutral-pH groundwater from Area 2 of the U.S. Department of Energy Field Research Center at Oak Ridge National Laboratory. Area 2 groundwater contains high concentrations of dissolved calcium (ca. 4 mM), and thus, release of phosphate during glycerol phosphate metabolism has the potential to create conditions favorable for U(VI) sequestration in Ca-P minerals. Microbial enumeration and isolation studies verified the presence of aerobic and nitrate-reducing glycerol 3-phosphate (G3P)-metabolizing microorganisms in Area 2 sediments. Coprecipitation of U(VI) with Ca-P minerals coupled to microbial G3P hydrolysis was demonstrated in artificial groundwater under aerobic and nitrate-reducing conditions. Transmission electron microscopy analysis and mineral-washing experiments demonstrated that U(VI) was incorporated into the structure of the insoluble Ca-P mineral hydroxyapatite [Ca₅(PO₄)₃OH]. Our results support the idea that U(VI) can be effectively removed from solution in contaminated aquifers through stimulation of microbial organophosphate metabolism.Reductive precipitation of U(VI) through stimulation of anaerobic, dissimilatory metal-reducing microbial activity is a demonstrated method for in situ immobilization of uranium in subsurface environments (3, 41). This is an attractive approach for aquifers that are naturally low in dissolved O₂ and NO₃⁻, e.g., the uranium-contaminated aquifer adjacent to the Colorado River in Rifle, CO (3). Alternative uranium remediation approaches may be called for in uranium-contaminated subsurface environments that contain significant concentrations of O₂ and NO₃⁻ entering through vertical recharge or lateral transport. One such potential strategy is immobilization of uranium through precipitation of U(VI)-bearing calcium-phosphate (Ca-P) phases (6, 39). Uranium incorporation into Ca-P minerals is a naturally occurring process. For example, U(VI)-phosphate phases at a weathered portion of the Coles Hill, VA, uranium ore deposit maintain groundwater U(VI) concentrations at ca. 0.06 μM (11, 12). The shallow portion of this deposit exhibits a sharp Fe redox front with U(IV) assemblages (containing uraninite, UO₂) located on the reduced side and U(VI) assemblages [containing autunite, Ca(UO₂)₂(PO₄)₃, and other uranium-bearing Ca-P phases] on the weathered oxidized side. Interestingly, uranium concentrations on both oxidized and reduced sides of the redox front are similar, suggesting no significant uranium loss as a result of oxidation and weathering.A major limitation for engineering uranium immobilization through coprecipitation with Ca-P minerals is the potential decrease in hydraulic conductivity of sediments due to rapid mineral precipitation at or near the point of P_i injection. In order to avoid this, P_i should be delivered into the subsurface at a relatively low rate. A potential amendment for in situ uranium remediation allowing slow P_i liberation is glycerol phosphate. Release of P_i from glycerol phosphate is promoted by microbial activity.Glycerol phosphate can facilitate microbial growth in two ways. First, it can serve as a P source for phosphatase-positive heterotrophic microorganisms. Phosphatases are enzymes that mediate hydrolysis of glycerol 3-phosphate (G3P) and other phosphate-containing compounds, resulting in a release of P_i for use in cell biosynthesis (33). Second, glycerol phosphate can serve as a combined source of phosphorus, carbon, and energy for growth by microorganisms. Such organisms may either carry out an initial extracellular glycerol phosphate hydrolysis step followed by glycerol and P_i uptake and utilization (33), or take up glycerol phosphate directly without hydrolysis in exchange for P_i (5, 16, 17). Phosphatase-positive organisms are often isolated from metal-contaminated sediments (13, 21, 25, 27, 31). One possible reason for this is that phosphatase activity decreases metal toxicity to the cells by causing precipitation of metal phosphate phases (13, 14, 24, 27, 28, 31, 35).Previous studies have demonstrated the efficacy of glycerol phosphate-hydrolyzing microorganisms in the removal of uranium and other radionuclides from solution through formation of metal phosphate mineral phases. Macaskie and coauthors (19, 20, 22-24) isolated a Citrobacter species strain from metal-contaminated soil that hydrolyzed G3P and accumulated heavy metals (as insoluble metal phosphates) on the cell wall. This finding served as a basis for proposed ex situ processes to remove uranium and other metals from contaminant waste streams. Beazley and colleagues (6) and Martinez et al. (25) determined the phosphatase activities of 135 strains isolated from acidic sediments at the U.S. Department of Energy Oak Ridge National Laboratory Field Research Center (ORFRC) and demonstrated that activity of these bacteria can promote uranium immobilization through formation of autunite/meta-autunite group minerals.The purpose of this study was to evaluate the potential for in situ immobilization of U(VI) in Area 2 subsurface sediments at the ORFRC. The circumneutral-pH groundwater at Area 2 contains high concentrations of dissolved calcium (ca. 4 mM). Hence, release of P_i during microbial G3P metabolism has the potential to create conditions favorable for Ca-P mineral precipitation and U(VI) sequestration. The key questions examined were the abundance of G3P-metabolizing microorganisms in Area 2 sediments and the nature and stability of the uranium-bearing Ca-P precipitates formed during microbial G3P metabolism. G3P-metabolizing organisms, capable of utilizing G3P as a combined phosphorus, carbon, and energy source, are likely to be preferentially stimulated in organic-matter-poor subsurface sediments, such as those at the ORFRC. G3P was therefore provided as the sole carbon and energy source in all our experiments.     

Multivalent Human Papillomavirus L1 DNA Vaccination Utilizing Electroporation

Objectives

Naked DNA vaccines can be manufactured simply and are stable at ambient temperature, but require improved delivery technologies to boost immunogenicity. Here we explore in vivo electroporation for multivalent codon-optimized human papillomavirus (HPV) L1 and L2 DNA vaccination.

Methods

Balb/c mice were vaccinated three times at two week intervals with a fusion protein comprising L2 residues ∼11−88 of 8 different HPV types (11−88×8) or its DNA expression vector, DNA constructs expressing L1 only or L1+L2 of a single HPV type, or as a mixture of several high-risk HPV types and administered utilizing electroporation, i.m. injection or gene gun. Serum was collected two weeks and 3 months after the last vaccination. Sera from immunized mice were tested for in-vitro neutralization titer, and protective efficacy upon passive transfer to naive mice and vaginal HPV challenge. Heterotypic interactions between L1 proteins of HPV6, HPV16 and HPV18 in 293TT cells were tested by co-precipitation using type-specific monoclonal antibodies.

Results

Electroporation with L2 multimer DNA did not elicit detectable antibody titer, whereas DNA expressing L1 or L1+L2 induced L1-specific, type-restricted neutralizing antibodies, with titers approaching those induced by Gardasil. Co-expression of L2 neither augmented L1-specific responses nor induced L2-specific antibodies. Delivery of HPV L1 DNA via in vivo electroporation produces a stronger antibody response compared to i.m. injection or i.d. ballistic delivery via gene gun. Reduced neutralizing antibody titers were observed for certain types when vaccinating with a mixture of L1 (or L1+L2) vectors of multiple HPV types, likely resulting from heterotypic L1 interactions observed in co-immunoprecipitation studies. High titers were restored by vaccinating with individual constructs at different sites, or partially recovered by co-expression of L2, such that durable protective antibody titers were achieved for each type.

Discussion

Multivalent vaccination via in vivo electroporation requires spatial separation of individual type L1 DNA vaccines.     

Persistence, extinction and different species pools within the flora of lake islands in western Ireland

The vascular flora on twenty-nine lake islands in Lough Corrib, western Ireland was surveyed in 1992–93. Thirteen of these islands had been surveyed by the author in 1974 (Roden, 1979). Data on species–area curves and species turnover between 1974 and 1992 are presented. Species numbers on each island did not change greatly in the 18-year interval and extinctions were most common on smaller islands. It is known that six of the islands surveyed are less than 150 years old and their flora must have immigrated over open water during that period. It is shown that this group of species has a different log species/log area regression than the remaining flora, with a much shallower slope (low Z value). The proportion of less widespread species was greatest on islands nearest to the mainland. The implication of different slopes in different species groups and the restriction of turnover to rare species is discussed with reference to the island Theory of Biogeography.     

Picking out parallels: plant circadian clocks in context.

Molecular models have been described for the circadian clocks of representatives of several different taxa. Much of the work on the plant circadian system has been carried out using the thale cress, Arabidopsis thaliana, as a model. We discuss the roles of genes implicated in the plant circadian system, with special emphasis on Arabidopsis. Plants have an endogenous clock that regulates many aspects of circadian and photoperiodic behaviour. Despite the discovery of components that resemble those involved in the clocks of animals or fungi, no coherent model of the plant clock has yet been proposed. In this review, we aim to provide an overview of studies of the Arabidopsis circadian system. We shall compare these with results from different taxa and discuss them in the context of what is known about clocks in other organisms.