The type II secretion system (Xcp) of Pseudomonas putida is active and involved in the secretion of phosphatases.

The genome of the Gram-negative bacterium Pseudomonas putida harbours a complete set of xcp genes for a type II protein secretion system (T2SS). This study shows that expression of these genes is induced under inorganic phosphate (Pi ) limitation and that the system enables the utilization of various organic phosphate sources. A phosphatase of the PhoX family, previously designated UxpB, was identified, which was produced under low Pi conditions and transported across the cell envelope in an Xcp-dependent manner demonstrating that the xcp genes encode an active T2SS. The signal sequence of UxpB contains a twin-arginine translocation (Tat) motif as well as a lipobox, and both processing by leader peptidase II and Tat dependency were experimentally confirmed. Two different tat gene clusters were detected in the P.?putida genome, of which one, named tat-1, is located adjacent to the uxpB and xcp genes. Both Tat systems appeared to be capable of transporting the UxpB protein. However, expression of the tat-1 genes was strongly induced by low Pi levels, indicating a function of this system in survival during Pi starvation.

The type II secretion system (Xcp) of Pseudomonas putida is active and involved in the secretion of phosphatases.

The genome of the Gram-negative bacterium Pseudomonas putida harbours a complete set of xcp genes for a type II protein secretion system (T2SS). This study shows that expression of these genes is induced under inorganic phosphate (Pi ) limitation and that the system enables the utilization of various organic phosphate sources. A phosphatase of the PhoX family, previously designated UxpB, was identified, which was produced under low Pi conditions and transported across the cell envelope in an Xcp-dependent manner demonstrating that the xcp genes encode an active T2SS. The signal sequence of UxpB contains a twin-arginine translocation (Tat) motif as well as a lipobox, and both processing by leader peptidase II and Tat dependency were experimentally confirmed. Two different tat gene clusters were detected in the P.?putida genome, of which one, named tat-1, is located adjacent to the uxpB and xcp genes. Both Tat systems appeared to be capable of transporting the UxpB protein. However, expression of the tat-1 genes was strongly induced by low Pi levels, indicating a function of this system in survival during Pi starvation.

Purification and characterization of acetone carboxylase from Xanthobacter strain Py2

Acetone metabolism in the aerobic bacterium Xanthobacter strain Py2 proceeds by a carboxylation reaction forming acetoacetate as the first detectable product. In this study, acetone carboxylase, the enzyme catalyzing this reaction, has been purified to homogeneity and characterized. Acetone carboxylase was comprised of three polypeptides with molecular weights of 85,300, 78,300, and 19,600 arranged in an α2β2γ2 quaternary structure. The carboxylation of acetone was coupled to the hydrolysis of ATP and formation of 1 mol AMP and 2 mol inorganic phosphate per mol acetoacetate formed. ADP was also formed during the course of acetone consumption, but only accumulated at low, substoichiometric levels (≈10% yield) relative to acetoacetate. Inorganic pyrophosphate could not be detected as an intermediate or product of acetone carboxylation. In the absence of CO2, acetone carboxylase catalyzed the acetone-dependent hydrolysis of ATP to form both ADP and AMP, with ADP accumulating to higher levels than AMP during the course of the assays. Acetone carboxylase did not have inorganic pyrophosphatase activity. Acetone carboxylase exhibited a Vmax for acetone carboxylation of 0.225 μmol acetoacetate formed min−1⋅mg−1 at 30°C and pH 7.6 and apparent Km values of 7.80 μM (acetone), 122 μM (ATP), and 4.17 mM (CO2 plus bicarbonate). These studies reveal molecular properties of the first bacterial acetone-metabolizing enzyme to be isolated and suggest a novel mechanism of acetone carboxylation coupled to ATP hydrolysis and AMP and inorganic phosphate formation.

Characterization of Schizosaccharomyces pombe RNA triphosphatase

RNA triphosphatase catalyzes the first step in mRNA cap formation which entails the cleavage of the β–γ phosphoanhydride bond of triphosphate-terminated RNA to yield a diphosphate end that is then capped with GMP by RNA guanylyltransferase. Here we characterize a 303 amino acid RNA triphosphatase (Pct1p) encoded by the fission yeast Schizosaccharomyces pombe. Pct1p hydrolyzes the γ phosphate of triphosphate-terminated poly(A) in the presence of magnesium. Pct1p also hydrolyzes ATP to ADP and Pi in the presence of manganese or cobalt (Km = 19 µM ATP; kcat = 67 s–1). Hydrolysis of 1 mM ATP is inhibited with increasing potency by inorganic phosphate (I0.5 = 1 mM), pyrophosphate (I0.5 = 0.4 mM) and tripolyphosphate (I0.5 = 30 µM). Velocity sedimentation indicates that Pct1p is a homodimer. Pct1p is biochemically and structurally similar to the catalytic domain of Saccharomyces cerevisiae RNA triphosphatase Cet1p. Mechanistic conservation between Pct1p and Cet1p is underscored by a mutational analysis of the putative metal-binding site of Pct1p. Pct1p is functional in vivo in S.cerevisiae in lieu of Cet1p, provided that it is coexpressed with the S.pombe guanylyltransferase. Pct1p and other yeast RNA triphosphatases are completely unrelated, mechanistically and structurally, to the metazoan RNA triphosphatases, suggesting an abrupt evolutionary divergence of the capping apparatus during the transition from fungal to metazoan species.

Organellar and Cytosolic Localization of Four Phosphoribosyl Diphosphate Synthase Isozymes in Spinach

Four cDNAs encoding phosphoribosyl diphosphate (PRPP) synthase were isolated from a spinach (Spinacia oleracea) cDNA library by complementation of an Escherichia coli Δprs mutation. The four gene products produced PRPP in vitro from ATP and ribose-5-phosphate. Two of the enzymes (isozymes 1 and 2) required inorganic phosphate for activity, whereas the others were phosphate independent. PRPP synthase isozymes 2 and 3 contained 76 and 87 amino acid extensions, respectively, at their N-terminal ends in comparison with other PRPP synthases. Isozyme 2 was synthesized in vitro and shown to be imported and processed by pea (Pisum sativum) chloroplasts. Amino acid sequence analysis indicated that isozyme 3 may be transported to mitochondria and that isozyme 4 may be located in the cytosol. The deduced amino acid sequences of isozymes 1 and 2 and isozymes 3 and 4 were 88% and 75% identical, respectively. In contrast, the amino acid identities of PRPP synthase isozyme 1 or 2 with 3 or 4 was modest (22%–25%), but the sequence motifs for binding of PRPP and divalent cation-nucleotide were identified in all four sequences. The results indicate that PRPP synthase isozymes 3 and 4 belong to a new class of PRPP synthases that may be specific to plants.

Evidence for a Slow-Turnover Form of the Ca2+-Independent Phosphoenolpyruvate Carboxylase Kinase in the Aleurone-Endosperm Tissue of Germinating Barley Seeds1

Phosphoenolpyruvate carboxylase (PEPC) activity was detected in aleurone-endosperm extracts of barley (Hordeum vulgare) seeds during germination, and specific anti-sorghum (Sorghum bicolor) C4 PEPC polyclonal antibodies immunodecorated constitutive 103-kD and inducible 108-kD PEPC polypeptides in western analysis. The 103- and 108-kD polypeptides were radiolabeled in situ after imbibition for up to 1.5 d in 32P-labeled inorganic phosphate. In vitro phosphorylation by a Ca2+-independent PEPC protein kinase (PK) in crude extracts enhanced the enzyme's velocity and decreased its sensitivity to l-malate at suboptimal pH and [PEP]. Isolated aleurone cell protoplasts contained both phosphorylated PEPC and a Ca2+-independent PEPC-PK that was partially purified by affinity chromatography on blue dextran-agarose. This PK activity was present in dry seeds, and PEPC phosphorylation in situ during imbibition was not affected by the cytosolic protein-synthesis inhibitor cycloheximide, by weak acids, or by various pharmacological reagents that had proven to be effective blockers of the light signal transduction chain and PEPC phosphorylation in C4 mesophyll protoplasts. These collective data support the hypothesis that this Ca2+-independent PEPC-PK was formed during maturation of barley seeds and that its presumed underlying signaling elements were no longer operative during germination.

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Content, Assimilatory Charge, and Mesophyll Conductance in Leaves1

The content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (Et; EC 4.1.1.39) measured in different-aged leaves of sunflower (Helianthus annuus) and other plants grown under different light intensities, varied from 2 to 75 μmol active sites m−2. Mesophyll conductance (μ) was measured under 1.5% O2, as well as postillumination CO2 uptake (assimilatory charge, a gas-exchange measure of the ribulose-1,5-bisphosphate pool). The dependence of μ on Et saturated at Et = 30 μmol active sites m−2 and μ = 11 mm s−1 in high-light-grown leaves. In low-light-grown leaves the dependence tended toward saturation at similar Et but reached a μ of only 6 to 8 mm s−1. μ was proportional to the assimilatory charge, with the proportionality constant (specific carboxylation efficiency) between 0.04 and 0.075 μm−1 s−1. Our data show that the saturation of the relationship between Et and μ is caused by three limiting components: (a) the physical diffusion resistance (a minor limitation), (b) less than full activation of Rubisco (related to Rubisco activase and the slower diffusibility of Rubisco at high protein concentrations in the stroma), and (c) chloroplast metabolites, especially 3-phosphoglyceric acid and free inorganic phosphate, which control the reaction kinetics of ribulose-1,5-bisphosphate carboxylation by competitive binding to active sites.

Regulation of S-Like Ribonuclease Levels in Arabidopsis. Antisense Inhibition of RNS1 or RNS2 Elevates Anthocyanin Accumulation1

The S-like ribonucleases (RNases) RNS1 and RNS2 of Arabidopsis are members of the widespread T2 ribonuclease family, whose members also include the S-RNases, involved in gametophytic self-incompatibility in plants. Both RNS1 and RNS2 mRNAs have been shown previously to be induced by inorganic phosphate (Pi) starvation. In our study we examined this regulation at the protein level and determined the effects of diminishing RNS1 and RNS2 expression using antisense techniques. The Pi-starvation control of RNS1 and RNS2 was confirmed using antibodies specific for each protein. These specific antibodies also demonstrated that RNS1 is secreted, whereas RNS2 is intracellular. By introducing antisense constructs, mRNA accumulation was inhibited by up to 90% for RNS1 and up to 65% for RNS2. These plants contained abnormally high levels of anthocyanins, the production of which is often associated with several forms of stress, including Pi starvation. This effect demonstrates that diminishing the amounts of either RNS1 or RNS2 leads to effects that cannot be compensated for by the actions of other RNases, even though Arabidopsis contains a large number of different RNase activities. These results, together with the differential localization of the proteins, imply that RNS1 and RNS2 have distinct functions in the plant.

Phosphorylated Nitrate Reductase and 14-3-3 Proteins1 : Site of Interaction, Effects of Ions, and Evidence for an AMP-Binding Site on 14-3-3 Proteins

The inactivation of phosphorylated nitrate reductase (NR) by the binding of 14-3-3 proteins is one of a very few unambiguous biological functions for 14-3-3 proteins. We report here that serine and threonine residues at the +6 to +8 positions, relative to the known regulatory binding site involving serine-543, are important in the interaction with GF14ω, a recombinant plant 14-3-3. Also shown is that an increase in ionic strength with KCl or inorganic phosphate, known physical effectors of NR activity, directly disrupts the binding of protein and peptide ligands to 14-3-3 proteins. Increased ionic strength attributable to KCl caused a change in conformation of GF14ω, resulting in reduced surface hydrophobicity, as visualized with a fluorescent probe. Similarly, it is shown that the 5′ isomer of AMP was specifically able to disrupt the inactive phosphorylated NR:14-3-3 complex. Using the 5′-AMP fluorescent analog trinitrophenyl-AMP, we show that there is a probable AMP-binding site on GF14ω.

Metabolism of Uridine 5′-Diphosphate-Glucose in Golgi Vesicles from Pea Stems1

Uridine 5′-diphosphate-glucose (UDP-Glc) is transported into the lumen of the Golgi cisternae, where is used for polysaccharide biosynthesis. When Golgi vesicles were incubated with UDP-[3H]Glc, [3H]Glc was rapidly transferred to endogenous acceptors and UDP-Glc was undetectable in Golgi vesicles. This result indicated that a uridine-containing nucleotide was rapidly formed in the Golgi vesicles. Since little is known about the fate of the nucleotide derived from UDP-Glc, we analyzed the metabolism of the nucleotide moiety of UDP-Glc by incubating Golgi vesicles with [α-32P]UDP-Glc, [β-32P]UDP-Glc, and [3H]UDP-Glc and identifying the resulting products. After incubation of Golgi vesicles with these radiolabeled substrates we could detect only uridine 5′-monophosphate (UMP) and inorganic phosphate (Pi). UDP could not be detected, suggesting a rapid hydrolysis of UDP by the Golgi UDPase. The by-products of UDP hydrolysis, UMP and Pi, did not accumulate in the lumen, indicating that they were able to exit the Golgi lumen. The exit of UMP was stimulated by UDP-Glc, suggesting the presence of a putative UDP-Glc/UMP antiporter in the Golgi membrane. However, the exit of Pi was not stimulated by UDP-Glc, suggesting that the exit of Pi occurs via an independent membrane transporter.

Reversibility of H+-ATPase and H+-Pyrophosphatase in Tonoplast Vesicles from Maize Coleoptiles and Seeds1

Tonoplast-enriched vesicles isolated from maize (Zea mays L.) coleoptiles and seeds synthesize ATP from ADP and inorganic phosphate (Pi) and inorganic pyrophosphate from Pi. The synthesis is consistent with reversal of the catalytic cycle of the H+-ATPase and H+-pyrophosphatase (PPase) vacuolar membrane-bound enzymes. This was monitored by measuring the exchange reaction that leads to 32Pi incorporation into ATP or inorganic pyrophosphate. The reversal reactions of these enzymes were dependent on the proton gradient formed across the vesicle membrane and were susceptible to the uncoupler carbonyl cyanide p(trifluoromethoxy)-phenylhydrazone and the detergent Triton X-100. Comparison of the two H+ pumps showed that the H+-ATPase was more active than H+-PPase in coleoptile tonoplast vesicles, whereas in seed vesicles H+-PPase activity was clearly dominant. These findings may reflect the physiological significance of these enzymes in different tissues at different stages of development and/or differentiation.

Caracterização limnológica do rio do Peixe (microrregião geográfica de Botucatu - SP), em duas épocas do ano (períodos de seca e chuva)

O rio do Peixe, um dos principais afluentes do rio Tietê (Reservatório de Barra Bonita/Hidrovia Tietê Paraná), juntamente com seus formadores (microrregião geográfica de Botucatu - SP) constituem os principais mananciais das cidades que se localizam em suas bacias. A maior parte da bacia hidrográfica do rio do Peixe desenvolve-se sob rochas sedimentares arenosas, favoráveis às atividades de extração de areia, fato que aliado ao manejo incorreto do solo favorece a ocorrência de processos erosivos. Foram realizadas amostragens de água durante dez dias consecutivos e de sedimento durante três dias alternados, em dois períodos hidrológicos (seca e chuva), para a determinação de variáveis físicas, químicas e biológicas,com o objetivo principal de caracterizar do ponto de vista limnológico esse rio e sua foz. As análises das diferentes variáveis seguiram os métodos que são utilizados rotineiramente no Laboratório de Limnologia do CRHEA/EESC/USP. As águas do rio do Peixe apresentaram, principalmente altas concentrações de sólidos em suspensão. Os maiores valores de turbidez, condutividade, alcalinidade, HCO3-, CO3-, nitrito, nitrogênio amoniacal, nitrogênio Kjedhal, fosfato inorgânico, carbono orgânico e sólidos em suspensão foram obtidos durante o período de chuvas, influenciados principalmente pelo escoamento superficial que promove o carreamento de materiais da bacia hidrográfica. Foi possível ainda através das características limnológicas, agrupar as estações de amostragem ao longo do contínuo do rio em três regiões (alto, médio e baixo curso), e diferenciar do ponto de vista trófico as estações da foz com o rio Tietê. Um aumento da biomassa fitoplanctônica, no período de seca, no baixo curso do rio do Peixe foi favorecido pela menor vazão na barragem de Barra Bonita.

Chemical observations in the Red Sea and the inner Gulf of Aden during the International Indian Ocean Expedition 1964/65

The Red Sea has a special place among the adjacent seas of the world. High evaporation, exclusion of its deep water from contact with the Indian Ocean proper and complete absence of continental drainage may result special conditions of the chemistry of the Red Sea. This paper aims to describe and explain the peculiarity of the hydrochemical situation. The influence of the topography, of the inflow and outflow through the straights of Bab el Mandeb, of the evaporation, of the stability of the water layers, and of the circulation will be studied. An attempt is made to estimate the apparent oxygen ultilisation in order to obtain an indication of the biological activity. A further attempt is made toward the quantitative estimation of the circulation of the nutrients and also to obtain some information about transport, dissolution, and precipitation of calcium carbonate. The basis of these investigations are mainly observations of R. V. "Meteor" during the International Indian Ocean Expedition 1964/65. The determination of dissolved oxygen, dissolved inorganic phosphate, nitrate, nitrite, ammonia, pH, alkalinity, silicate as well as salinity and temperature forms the necessary basis for such an investigation of the chemical conditions. In the first chapter the methods and some modifications for the determination of the chemical properties as applied during the I.I.O.E. cruise of R. V. "Meteor" are described. The new methods, as worked out and tested under sea going conditions during several years by the author, are described in more detail. These are the methods for nitrate, silicate, the automatic determination of dissolved inorganic phosphate and silicate, the automated determination of total phosphorus, the in situ recording of the oxygen tension, and the modification for the determination of ammonia, calcium, and dissolved oxygen. With these revised methods more than 18,000 determinations have been carried out during the Indian Ocean cruise. The complete working up of the chemical data of the Indian Ocean Expedition of R. V. "Meteor" is devided into four sections: Contributions 1) to the Chemistry of the Red Sea and the Inner Gulf of Aden, 2) to the Gulf of Aden and the Somali Coast Region, 3) to the Western Indian Coast Region, and 4) to the Persian Gulf and the Straits of Oman. This paper presents the first contribution. The special hydrographical conditions are discussed. It can be shown, that the increase of salinity in the surface waters from the south to the north of the Red Sea is only to about 30 % due to evaporation. The remaining increase is presumed to be due to the admixture of deep water to the surface layers. A special rate for the consumption of oxygen (0.114 ml/ l/a) is derived for the deep water of the Red Sea at 1500 m. Based upon the distribution of the dissolved oxygen along the axii of the Red Sea, a chematic model for the longitudinal circulation of the Red Sea is constructed. This model should be considered as a first approximation and may explain the special distribution of phosphate, nitrate, and silicate. Based upon the evaluation of the residence time of the deep water a dissolution rate for silicate is estimated as 1 mygat/a. It seems possible to calculate residence times of water masses outside the Red Sea from the silicate content. The increase of silicate and the consumption of oxygen lead to residence times of the water below the thermocine of 30 to 48 years. The distribution of oxygen in the Straits of Bab el Mandeb is described and discussed. The rate of consumption of the oxygen in the outflowing Red Sea water is estimated to 8.5 ml/ l/a. This rather high rate is explained with reference to the special conditions in the outflowing water. The Red Sea water is characterized initially by a relative high content of oxygen and a low content of nutrients. The increase in nutrients and the decrease in the oxygen content is a secondary process of the Red Sea water on its way to the Arabian Sea. Based upon the vertical distribution of the dissolved inorganic phosphate vertical exchange coefficients of 1 - 4 g/cm/sec and vertical current speeds of 10**-5 to 10**-4 cm/sec are calculated for some stations in the Red Sea. The distribution of phosphate, silicate, nitrate, nitrite and ammonia for the Red Sea and the Straits of Bab el Mandeb are discussed. The special circulation is evaluated and the balance of the nutrients is estimated by means of the brutto transport. The nutrient deficit is assumed to be balanced by sporadic inflow of intermediate water from the Gulf of Aden. An example for such an inflow has been observed and is demonstrated. The silicate-salinity relationships are a suitable way for characterizing water masses in the Red Sea. Equations for the calculation of the different components from the carbonate system, the ion activities, and the calcium carbonate saturation are evaluated. The influence of temperature and pressure is taken into account. The carbonate saturation is calculated from the determined concentrations of calcium, alkalinity, and the hydrogen ion activity. Saturation values of 320 % are found for the surface layer and of 100% ± 1 for the deep water. The extraordinary equilibrium conditions may explain the constant Ca/Cl ratio and also the sedimentation of undissolved carbonate skelecons even in greater depths. A main sedimentation rate of 2 * 10**-3cm/year is evaluated from a total sedimentation of 10 * 106 to/a of calcium carbonate in the Red Sea. The appendix contains those data, which are not published in the data volume of the I.I.O.E. expedition of R. V. "Meteor".

Competitive sorption reactions between phosphorus and organic matter in soil: a review

The incorporation of organic matter ( OM) in soils that are able to rapidly sorb applied phosphorus ( P) fertiliser reportedly increases P availability to plants. This effect has commonly been ascribed to competition between the decomposition products of OM and P for soil sorption sites resulting in increased soil solution P concentrations. The evidence for competitive inhibition of P sorption by dissolved organic carbon compounds, derived from the breakdown of OM, includes studies on the competition between P and (i) low molecular weight organic acids (LOAs), (ii) humic and fulvic acids, and (iii) OM leachates in soils with a high P sorption capacity. These studies, however, have often used LOAs at 1 - 100 mM, concentrations much higher than those in soils ( generally < 0.05 mM). The transience of LOAs in biologically active soils further suggests that neither their concentration nor their persistence would have a practical benefit in increasing P phytoavailability. Higher molecular weight compounds such as humic and fulvic acids also competitively inhibit P sorption; however, little consideration has been given to the potential of these compounds to increase the amount of P sorbed through metal - chelate linkages. We suggest that the magnitude of the inhibition of P sorption by the decomposition products of OM leachate is negligible at rates equivalent to those of OM applied in the field. Incubation of OM in soil has also commonly been reported as reducing P sorption in soil. However, we consider that the reported decreases in P sorption ( as measured by P in the soil solution) are not related to competition from the decomposition products of OM breakdown, but are the result of P release from the OM that was not accounted for when calculating the reduction in P sorption.