Plant growth-promoting bacteria that decrease heavy metal toxicity in plants

Kluyvera ascorbata SUD165 and a siderophore-overproducing mutant of this bacterium, K. ascorbata SUD165/26, were used to inoculate tomato, canola, and Indian mustard seeds which were then grown in soil for 25-42 days in the presence of either nickel, lead, or zinc. The parameters that were monitored included plant wet and dry weight, protein and chlorophyll content in the plant leaves, and concentration of heavy metal in the plant roots and shoots. As indicated by a decrease in the measured values of these parameters, in all instances, plant growth was inhibited by the presence of the added metal. Both bacterial strains were effective, although not always to a statistically significant extent, at relieving a portion of the growth inhibition caused by the metals. In most cases, the siderophore overproducing mutant K. ascorbata 165/26 exerted a more pronounced effect on plant growth than did the wild-type bacterium K. ascorbata SUD165. The data suggest that the ability of these bacteria to protect plants against the inhibitory effects of high concentrations of nickel, lead, and zinc is related to the bacteria providing the plants with sufficient iron.     

Biological activity and colonization pattern of the bioluminescence-labeled plant growth-promoting bacterium Kluyvera ascorbata SUD165/26

Kluyvera ascorbata SUD165/26 is a spontaneous siderophore-overproducing mutant of K. ascorbata SUD165, which was previously isolated from nickel-contaminated soil and shown to significantly enhance plant growth in soil contaminated with high levels of heavy metals. To develop a better understanding of the functioning of K. ascorbata SUD165/26 in the environment, and to trace its distribution in the rhizosphere, isolates of this bacterium were labeled with either green fluorescent protein or luciferase. When the plant growth-promoting activities of the labeled strains were assayed and compared with the activities of the unlabeled strain, none of the monitored parameters had changed to any significant extent. When the spatial colonization patterns of the labeled bacteria on canola roots were determined after seed application, it was observed that the bacterium was tightly attached to the surface of both roots and seeds, and formed aggregates. The majority of the bacterial population inhabited the upper two thirds of the roots, with no bacteria detected around the root tips.     

Three Mechanisms for the Calcium Alleviation of Mineral Toxicities

Ca²⁺ in rooting medium is essential for root elongation, even in the absence of added toxicants. In the presence of rhizotoxic levels of Al³⁺, H⁺, or Na⁺ (or other cationic toxicants), supplementation of the medium with higher levels of Ca²⁺ alleviates growth inhibition. Experiments to determine the mechanisms of alleviation entailed measurements of root elongation in wheat (Triticum aestivum L. cv Scout 66) seedlings in controlled medium. A Gouy-Chapman-Stern model was used to compute the electrical potentials and the activities of ions at the root-cell plasma membrane surfaces. Analysis of root elongation relative to the computed surface activities of ions revealed three separate mechanisms of Ca²⁺ alleviation. Mechanism I is the displacement of cell-surface toxicant by the Ca²⁺-induced reduction in cell-surface negativity. Mechanism II is the restoration of Ca²⁺ at the cell surface if the surface Ca²⁺ has been reduced by the toxicant to growth-limiting levels. Mechanism III is the collective ameliorative effect of Ca²⁺ beyond mechanisms I and II, and may involve Ca²⁺-toxicant interactions at the cell surface other than the displacement interactions of mechanisms I and II. Mechanism I operated in the alleviation of all of the tested toxicities; mechanism II was generally a minor component of alleviation; and mechanism III was toxicant specific and operated strongly in the alleviation of Na⁺ toxicity, moderately in the alleviation of H⁺ toxicity, and not at all in the alleviation of Al³⁺ toxicity.     

Uridine 5′-Diphosphate-Glucose Dehydrogenase from Soybean Nodules

A highly purified preparation of uridine 5′-diphosphate (UDP)-glucose (Glc) dehydrogenase (DH; EC 1.1.1.22) has been characterized from soybean (Glycine max L.) nodules. The enzyme had native and subunit molecular masses of approximately 272 and 50 kD, respectively. UDP-Glc DH displayed typical hyperbolic substrate kinetics and had K_m values for UDP-Glc and NAD⁺ of 0.05 and 0.12 mm, respectively. Thymidine 5′-diphosphate-Glc and UDP-galactose could replace UDP-Glc as the sugar nucleotide substrate to some extent, but the enzyme had no activity with NADP⁺. Soybean nodule UDP-Glc DH was labile in the absence of NAD⁺ and was inhibited by a heat-stable, low-molecular-mass solute in crude extracts of soybean nodules. UDP-Glc DH was also isolated from developing soybean seeds and shoots of 5-d-old wheat and canola seedlings and was shown to have similar affinities for UDP-Glc and NAD⁺ as those of the soybean nodule enzyme. UDP-Glc DH from all of these sources was most active in young, rapidly growing tissues.     

AtCCX3 is an Arabidopsis endomembrane H+ -dependent K+ transporter

The Arabidopsis (Arabidopsis thaliana) cation calcium exchangers (CCXs) were recently identified as a subfamily of cation transporters; however, no plant CCXs have been functionally characterized. Here, we show that Arabidopsis AtCCX3 (At3g14070) and AtCCX4 (At1g54115) can suppress yeast mutants defective in Na⁺, K⁺, and Mn²⁺ transport. We also report high-capacity uptake of ⁸⁶Rb⁺ in tonoplast-enriched vesicles from yeast expressing AtCCX3. Cation competition studies showed inhibition of ⁸⁶Rb⁺ uptake in AtCCX3 cells by excess Na⁺, K⁺, and Mn²⁺. Functional epitope-tagged AtCCX3 fusion proteins were localized to endomembranes in plants and yeast. In Arabidopsis, AtCCX3 is primarily expressed in flowers, while AtCCX4 is expressed throughout the plant. Quantitative polymerase chain reaction showed that expression of AtCCX3 increased in plants treated with NaCl, KCl, and MnCl₂. Insertional mutant lines of AtCCX3 and AtCCX4 displayed no apparent growth defects; however, overexpression of AtCCX3 caused increased Na⁺ accumulation and increased ⁸⁶Rb⁺ transport. Uptake of ⁸⁶Rb⁺ increased in tonoplast-enriched membranes isolated from Arabidopsis lines expressing CCX3 driven by the cauliflower mosaic virus 35S promoter. Overexpression of AtCCX3 in tobacco (Nicotiana tabacum) produced lesions in the leaves, stunted growth, and resulted in the accumulation of higher levels of numerous cations. In summary, these findings suggest that AtCCX3 is an endomembrane-localized H⁺-dependent K⁺ transporter with apparent Na⁺ and Mn²⁺ transport properties distinct from those of previously characterized plant transporters.     

Aluminum Induces a Decrease in Cytosolic Calcium Concentration in BY-2 Tobacco Cell Cultures

Al toxicity is a major problem that limits crop productivity on acid soils. It has been suggested that Al toxicity is linked to changes in cellular Ca homeostasis and the blockage of plasma membrane Ca²⁺-permeable channels. BY-2 suspension-cultured cells of tobacco (Nicotiana tabacum L.) exhibit rapid cell expansion that is sensitive to Al. Therefore, the effect of Al on changes in cytoplasmic free Ca concentration ([Ca²⁺]_cyt) was followed in BY-2 cells to assess whether Al perturbed cellular Ca homeostasis. Al exposure resulted in a prolonged reduction in [Ca²⁺]_cyt and inhibition of growth that was similar to the effect of the Ca²⁺ channel blocker La³⁺ and the Ca²⁺ chelator ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetraacetic acid. The Ca²⁺ channel blockers verapamil and nifedipine did not induce a decrease in [Ca²⁺]_cyt in these cells and also failed to inhibit growth. Al and La³⁺, but not verapamil or nifedipine, reduced the rate of Mn²⁺ quenching of Indo-1 fluorescence, which is consistent with the blockage of Ca²⁺- and Mn²⁺-permeable channels. These results suggest that Al may act to block Ca²⁺ channels at the plasma membrane of plant cells and this action may play a crucial role in the phytotoxic activity of the Al ion.     

Requirement of a Relatively High Threshold Level of Mg2+ for Cell Growth of a Rhizoplane Bacterium, Sphingomonas yanoikuyae EC-S001

Mg²⁺ is one of the essential elements for bacterial cell growth. The presence of the magnesium cation (Mg²⁺) in various concentrations often affects cell growth restoration in plant-associating bacteria. This study attempted to determine whether Mg²⁺ levels in Sphingomonas yanoikuyae EC-S001 affected cell growth restoration in the host plant and what the threshold level is. S. yanoikuyae EC-S001, isolated from the rhizoplane of spinach seedlings grown from surface-sterilized seeds under aseptic conditions, displayed uniform dispersion and attachment throughout the rhizoplane and phylloplane of the host seedlings. S. yanoikuyae EC-S001 did not grow in potato-dextrose broth medium but grew well in an aqueous extract of spinach leaves. Chemical investigation of the growth factor in the spinach leaf extract led to identification of the active principle as the magnesium cation. A concentration of ca. 0.10 mM Mg²⁺ or more allowed S. yanoikuyae EC-S001 to grow in potato-dextrose broth medium. Some saprophytic and/or diazotrophic bacteria used in our experiment were found to have diverse threshold levels for their Mg²⁺ requirements. For example, Burkholderia cepacia EC-K014, originally isolated from the rhizoplane of a Melastoma sp., could grow even in Mg²⁺-free Hoagland's no. 2 medium with saccharose and glutamine (HSG medium) and requires a trace level of Mg²⁺ for its growth. In contrast, S. yanoikuyae EC-S001, together with Bacillus subtilis IFO12113, showed the most drastic restoring responses to subsequent addition of 0.98 mM Mg²⁺ to Mg²⁺-free HSG medium. Our studies concluded that Mg²⁺ is more than just the essential trace element needed for cell growth restoration in S. yanoikuyae EC-S001 and that certain nonculturable bacteria may require a higher concentration of Mg²⁺ or another specific essential element for their growth.     

Properties of an Inwardly Rectifying ATP-sensitive K+ Channel in the Basolateral Membrane of Renal Proximal Tubule

The potassium conductance of the basolateral membrane (BLM) of proximal tubule cells is a critical regulator of transport since it is the major determinant of the negative cell membrane potential and is necessary for pump-leak coupling to the Na⁺,K⁺-ATPase pump. Despite this pivotal physiological role, the properties of this conductance have been incompletely characterized, in part due to difficulty gaining access to the BLM. We have investigated the properties of this BLM K⁺ conductance in dissociated, polarized Ambystoma proximal tubule cells. Nearly all seals made on Ambystoma cells contained inward rectifier K⁺ channels (γ_{slope, in} = 24.5 ± 0.6 pS, γ_{chord, out} = 3.7 ± 0.4 pS). The rectification is mediated in part by internal Mg²⁺. The open probability of the channel increases modestly with hyperpolarization. The inward conducting properties are described by a saturating binding–unbinding model. The channel conducts Tl⁺ and K⁺, but there is no significant conductance for Na⁺, Rb⁺, Cs⁺, Li⁺, NH₄⁺, or Cl⁻. The channel is inhibited by barium and the sulfonylurea agent glibenclamide, but not by tetraethylammonium. Channel rundown typically occurs in the absence of ATP, but cytosolic addition of 0.2 mM ATP (or any hydrolyzable nucleoside triphosphate) sustains channel activity indefinitely. Phosphorylation processes alone fail to sustain channel activity. Higher doses of ATP (or other nucleoside triphosphates) reversibly inhibit the channel. The K⁺ channel opener diazoxide opens the channel in the presence of 0.2 mM ATP, but does not alleviate the inhibition of millimolar doses of ATP. We conclude that this K⁺ channel is the major ATP-sensitive basolateral K⁺ conductance in the proximal tubule.     

The Adhesion-Associated sca Operon in Streptococcus gordonii Encodes an Inducible High-Affinity ABC Transporter for Mn2+ Uptake

ScaA lipoprotein in Streptococcus gordonii is a member of the LraI family of homologous polypeptides found among streptococci, pneumococci, and enterococci. It is the product of the third gene within the scaCBA operon encoding the components of an ATP-binding cassette (ABC) transporter system. Inactivation of scaC (ATP-binding protein) or scaA (substrate-binding protein) genes resulted in both impaired growth of cells and >70% inhibition of ⁵⁴Mn²⁺ uptake in media containing <0.5 μM Mn²⁺. In wild-type and scaC mutant cells, production of ScaA was induced at low concentrations of extracellular Mn²⁺ (<0.5 μM) and by the addition of ≥20 μM Zn²⁺. Sca permease-mediated uptake of ⁵⁴Mn²⁺ was inhibited by Zn²⁺ but not by Ca²⁺, Mg²⁺, Fe²⁺, or Cu²⁺. Reduced uptake of ⁵⁴Mn²⁺ by sca mutants and by wild-type cells in the presence of Zn²⁺ was abrogated by the uncoupler carbonylcyanide m-chlorophenylhydrazone, suggesting that Mn²⁺ uptake under these conditions was proton motive force dependent. The frequency of DNA-mediated transformation was reduced >20-fold in sca mutants. The addition of 0.1 mM Mn²⁺ to the transformation medium restored only partly the transformability of mutant cells, implying an alternate role for Sca proteins in the transformation process. Cells of sca mutants were unaffected in other binding properties tested and were unaffected in sensitivity to oxidants. The results show that Sca permease is a high-affinity mechanism for the acquisition of Mn²⁺ and is essential for growth of streptococci under Mn²⁺-limiting conditions.     

YscO of Yersinia pestis Is a Mobile Core Component of the Yop Secretion System

The Yersinia pestis low-Ca²⁺ response stimulon is responsible for the temperature- and Ca²⁺-regulated expression and secretion of plasmid pCD1-encoded antihost proteins (V antigen and Yops). We have previously shown that lcrD, yscC, yscD, yscG, and yscR encode proteins that are essential for high-level expression and secretion of V antigen and Yops at 37°C in the absence of Ca²⁺. In this study, we characterized yscO of the Yop secretion (ysc) operon that contains yscN through yscU by determining the localization of its gene product and the phenotype of an in-frame deletion. The yscO mutant grew and expressed the same levels of Yops as the parent at 37°C in the presence of Ca²⁺. In the absence of Ca²⁺, the mutant grew independently of Ca²⁺, expressed only basal levels of V antigen and Yops, and failed to secrete these. These defects could be partially complemented by providing yscO in trans in the yscO mutant. Overexpression of YopM and V antigen in the mutant failed to restore the export of either protein, showing that the mutation had a direct effect on secretion. These results indicated that the yscO gene product is required for high-level expression and secretion of V antigen and Yops. YscO was found by immunoblot analysis in the soluble and membrane fractions of bacteria growing at 37°C irrespective of the presence of Ca²⁺ and in the culture medium in the absence of Ca²⁺. YscO is the only mobile protein identified so far in the Yersinia species that is required for secretion of V antigen and Yops.