Ions serve as essential nutrients in higher plants and can also act as signaling molecules. Little is known about how plants sense changes in soil nutrient concentrations. Previous studies showed that T101-phosphorylated CHL1 is a high-affinity nitrate transporter, whereas T101-dephosphorylated CHL1 is a low-affinity transporter. In this study, analysis of an uptake- and sensing-decoupled mutant showed that the nitrate transporter CHL1 functions as a nitrate sensor. Primary nitrate responses in CHL1T101D and CHLT101A transgenic plants showed that phosphorylated and dephosphorylated CHL1 lead to a low- and high-level response, respectively. In vitro and in vivo studies showed that, in response to low nitrate concentrations, protein kinase CIPK23 can phosphorylate T101 of CHL1 to maintain a low-level primary response. Thus, CHL1 uses dual-affinity binding and a phosphorylation switch to sense a wide range of nitrate concentrations in the soil, thereby functioning as an ion sensor in higher plants. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.

37Salmonella strains have recently been developed as antitumor agents capable of both preferentially amplifying within tumors and expressing prodrug-converting enzymes such as the herpes simplex thymidine kinase 1 . These bacteria were attenuated by auxotrophic mutations that limited their pathogenesis in normal tissues but retained high-level replication within the tumors following systemic administration. The auxotrophic requirements of these Salmonella are apparently met within the tumor environment where they then replicate, reaching up to more than 1000 times the concentration found in normal tissues.A significant limitation for safe use of systemically administered bacteria in humans is the ability of the bacteria to induce tumor necrosis factor α (TNFα)-mediated septic shock 2,3 . However, modifications in bacterial components responsible for eliciting host immune responses such as TNFα induction could interfere with tumor targeting or antitumor activity.Several mutations in lipid biosynthesis are known in Escherichia coli and Salmonella sp. that lower TNFα induction and render the bacteria nontoxic. Some mutations, such as kdo -result in the production of lipid IV A , which substantially lowers TNFα induction and acts as an antagonist to the TNFα response from wild-type lipid A 4,5 . However, these and most other lipid mutations are temperature-sensitive and conditionally lethal to the bacteria 6 , limiting the potential for tumor-based amplification seen in auxotrophic Salmonella 1 .In E. coli, the msbB (mlt) gene 7,8 is involved in the terminal myristoylation of lipid A 9,10 . Genetic disruption of this gene in E. coli results in a stable nonconditional mutation that lowers TNFα induction up to 10-fold by whole bacteria or up to 10,000-fold by purified lipopolysaccharide (LPS) 9 . A similar toxicity profile is observed when the msbB gene is disrupted in Salmonella 11 . We generated a deletion in the coding sequence of msbB within a hyperinvasive strain of Salmonella we previously used for tumor-targeting as well as the parental wild type, and examined the effect on virulence and TNFα production both in vitro and in vivo. Results indicate that msbB -mutant Salmonella retain the properties of tumor accumulation and tumor suppression in the absence of eliciting high levels of TNFα. Results Isolation and genetic disruption of the Salmonella msbB gene.DNA sequence analysis of Salmonella msbB clones obtained by DNA/DNA hybridization indicated the presence of an msbB homolog with flanking gene organization (orfU, msbB, pykA, and zwf) identical to E. coli 8 . The DNA homology of the Salmonella msbB and the E. coli msbB was determined to be 75%, and the amino acid homology 98%, confirming that the cloned Salmonella gene is an msbB homolog.Putative knockouts obtained by transformation of the linearized deletion construct were confirmed by several criteria using Southern blot analysis (Fig. 1): Two bands corresponding to the tetracycline gene were observed in the knockout construct and in the knockout clones and w...

Little is known about the molecular and regulatory mechanisms of long-distance nitrate transport in higher plants. NRT1.5 is one of the 53 Arabidopsis thaliana nitrate transporter NRT1 (Peptide Transporter PTR) genes, of which two members, NRT1.1 (CHL1 for Chlorate resistant 1) and NRT1.2, have been shown to be involved in nitrate uptake. Functional analysis of cRNA-injected Xenopus laevis oocytes showed that NRT1.5 is a low-affinity, pH-dependent bidirectional nitrate transporter. Subcellular localization in plant protoplasts and in planta promoter-b-glucuronidase analysis, as well as in situ hybridization, showed that NRT1.5 is located in the plasma membrane and is expressed in root pericycle cells close to the xylem. Knockdown or knockout mutations of NRT1.5 reduced the amount of nitrate transported from the root to the shoot, suggesting that NRT1.5 participates in root xylem loading of nitrate. However, root-to-shoot nitrate transport was not completely eliminated in the NRT1.5 knockout mutant, and reduction of NRT1.5 in the nrt1.1 background did not affect rootto-shoot nitrate transport. These data suggest that, in addition to that involving NRT1.5, another mechanism is responsible for xylem loading of nitrate. Further analyses of the nrt1.5 mutants revealed a regulatory loop between nitrate and potassium at the xylem transport step. INTRODUCTIONNitrate and ammonium ions are the two major nitrogen sources for higher plants. Due to its toxicity, ammonium is preferentially assimilated in the root and then transported in an organic form to the aerial parts. By contrast, nitrate can be assimilated into ammonium and then amino acids in the root or shoot. Partitioning of nitrate assimilation between the root and shoot depends on the plant species, external nitrate concentration, temperature, and light intensity (reviewed in Smirnoff and Stewart, 1985). If there is sufficient light, nitrate assimilation in the leaf has a lower energy cost than in the root. However, some disadvantages of leaf nitrate assimilation include (1) if light is limited, nitrate assimilation and carbon dioxide fixation will compete directly for the reductants and ATP generated by photosynthetic electron transport (Canvin and Atkins, 1974), and (2) hydroxyl ions generated in the leaf need to be neutralized by the synthesis of organic acids (in the root, the pH balance may possibly be maintained by reducing proton excretion or increasing bicarbonate excretion). Due to these factors, the partition of nitrate assimilation between the root and shoot shows both seasonal and diurnal fluctuations, allowing the plant to sustain maximal growth. In turn, the partition of nitrate assimilation depends on the partition of nitrate between the root and shoot.To transport nitrate to the aerial parts of the plant, nitrate has to be loaded into the xylem vessels of the root vascular stele. In Arabidopsis thaliana roots, four layers of cells are found surrounding the xylem, these being the epidermis, cortex, endodermis, and pericycle (in the order external to ...

Two-thirds of the lipid A in wild-type-component regulatory system, and also occurred in E. coli msbB or htrB mutants. The lipid A variants that accumulate in NH 4 VO 3 -treated E. coli K12 are the same as many of those normally found in untreated Salmonella typhimurium and Salmonella minnesota, demonstrating that E. coli K12 has latent enzyme systems for synthesizing these important derivatives.