SUMMARY After several decades of use of glyphosate, the active ingredient in weed killers such as Roundup, in fields, forests, and gardens, the biochemical pathway of transformation of glyphosate phosphorus to a useful phosphorus source for microorganisms has been disclosed. Glyphosate is a member of a large group of chemicals, phosphonic acids or phosphonates, which are characterized by a carbon-phosphorus bond. This is in contrast to the general phosphorus compounds utilized and metabolized by microorganisms. Here phosphorus is found as phosphoric acid or phosphate ion, phosphoric acid esters, or phosphoric acid anhydrides. The latter compounds contain phosphorus that is bound only to oxygen. Hydrolytic, oxidative, and radical-based mechanisms for carbon-phosphorus bond cleavage have been described. This review deals with the radical-based mechanism employed by the carbon-phosphorus lyase of the carbon-phosphorus lyase pathway, which involves reactions for activation of phosphonate, carbon-phosphorus bond cleavage, and further chemical transformation before a useful phosphate ion is generated in a series of seven or eight enzyme-catalyzed reactions. The phn genes, encoding the enzymes for this pathway, are widespread among bacterial species. The processes are described with emphasis on glyphosate as a substrate. Additionally, the catabolism of glyphosate is intimately connected with that of aminomethylphosphonate, which is also treated in this review. Results of physiological and genetic analyses are combined with those of bioinformatics analyses.

We have characterized the development of seeds in the model legume Lotus japonicus. Like soybean (Glycine max) and pea (Pisum sativum), Lotus develops straight seed pods and each pod contains approximately 20 seeds that reach maturity within 40 days. Histological sections show the characteristic three developmental phases of legume seeds and the presence of embryo, endosperm, and seed coat in desiccated seeds. Furthermore, protein, oil, starch, phytic acid, and ash contents were determined, and this indicates that the composition of mature Lotus seed is more similar to soybean than to pea. In a first attempt to determine the seed proteome, both a two-dimensional polyacrylamide gel electrophoresis approach and a gel-based liquid chromatography-mass spectrometry approach were used. Globulins were analyzed by two-dimensional polyacrylamide gel electrophoresis, and five legumins, LLP1 to LLP5, and two convicilins, LCP1 and LCP2, were identified by matrix-assisted laser desorption ionization quadrupole/time-of-flight mass spectrometry. For two distinct developmental phases, seed filling and desiccation, a gel-based liquid chromatography-mass spectrometry approach was used, and 665 and 181 unique proteins corresponding to gene accession numbers were identified for the two phases, respectively. All of the proteome data, including the experimental data and mass spectrometry spectra peaks, were collected in a database that is available to the scientific community via a Web interface (http://www.cbs.dtu.dk/cgi-bin/lotus/db.cgi). This database establishes the basis for relating physiology, biochemistry, and regulation of seed development in Lotus. Together with a new Web interface (http:// bioinfoserver.rsbs.anu.edu.au/utils/PathExpress4legumes/) collecting all protein identifications for Lotus, Medicago, and soybean seed proteomes, this database is a valuable resource for comparative seed proteomics and pathway analysis within and beyond the legume family.

Genes coding for enzymes functioning in purine salvage pathways have been located on the chromosome of Escherichia coli. The gene add encoding adenosine deaminase was located by transduction at 31 min, the gene order was established to be man-uidA-add-aroD. A deletion covering man-uidA-add was obtained. The gene gsk encoding guanosine kinase was cotransducible with purE and shown to be located at 13 min. The gene hpt encoding hypoxanthine phosphoribosyltransferase was cotransducible with tonA indicating a location at 3 min. The location of the gene gpt encoding guanine (xanthine) phosphoribosyltransferase in the proA-proB region was confirmed.

The nucleotide sequence of a 3.1 kb segment carrying the cytosine deaminase gene (codA) from Escherichia coli was determined. The sequence revealed the presence of two open reading frames, the first (codB) specifying a highly hydrophobic polypeptide and the second specifying cytosine deaminase. A two-codon overlap between the two reading frames indicates that they constitute an operon. Transcription of the operon was found to be regulated by exogenous purines. Polypeptides specified by each of the two reading frames were expressed in minicells, and the codB gene product was found to be highly enriched in the membrane fraction. Uptake experiments showed that the CodB protein is required for cytosine transport into the cell and that the intracellular accumulation of cytosine correlated with the codB gene dose. A topological model for the cytosine permease in the cytoplasmic membrane is proposed.

SummaryPhosphorous is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use organic phosphonate compounds, which require specialised enzymatic machinery for breaking the stable carbon-phosphorus (C-P) bond. Despite its importance, the details of how this machinery catabolises phosphonate remain unknown. Here we determine the crystal structure of the 240 kDa Escherichia coli C-P lyase core complex (PhnGHIJ) and show that it is a two-fold symmetric hetero-octamer comprising an intertwined network of subunits with unexpected selfhomologies. It contains two potential active sites that likely couple organic phosphonate compounds to ATP and subsequently hydrolyse the C-P bond. We map the binding site of PhnK on the complex using electron microscopy and show that it binds to PhnJ via a conserved insertion domain. Our results provide a structural basis for understanding microbial phosphonate breakdown.Phosphonate compounds that contain a stable carbon-phosphorus (C-P) bond are utilised as a source of phosphate by microorganisms in many natural environments where the low levels of free and organic phosphate limit growth 1 . The C-P lyase pathway, which converts phosphonate into 5-phosphoribosyl-α-1-diphosphate (PRPP) in an ATP-dependent fashion, is activated upon phosphate starvation in many bacterial species including Escherichia coli 2,3 . The enzymes of this pathway have a very broad substrate specificity enabling the Reprints and permissions information is available at www.nature.com/reprintsUsers may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http:// www.nature.com/authors/editorial_policies/license.html#terms 3 Correspondence and requests for materials should be addressed to D.E.B.(deb@mbg.au.dk, phone +45 21669001). Author Contributions. P.S., L.A.P., B.H.J., B.J., and D.E.B. designed and P.S., L.B.V., C.J.R, and B.J. carried out the experiments. P.S., M.K., and D.E.B. determined the crystal and EM structures while C.J.R. and L.A.P. carried out final refinement of the EM structure as well as EM structure validation. P.S, M.K., C.J.R., L.A.P., B.H.J., B.J., and D.E.B. wrote the manuscript.Atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) with accession code 4XB6. The EM density map has been deposited in the Electron Microscopy Data Bank (EMDB) with accession code EMD-3033.