Bjarne Jochimsen

The proteome of seed development in the model legume Lotus japonicus

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.

Five phosphonate operon gene products as components of a multi-subunit complex of the carbon-phosphorus lyase pathway

Organophosphonate utilization by Escherichia coli requires the 14 cistrons of the phnCDEFGHIJKLMNOP operon, of which the carbon-phosphorus lyase has been postulated to consist of the seven polypeptides specified by phnG to phnM. A 5,660-bp DNA fragment encompassing phnGHIJKLM is cloned, followed by expression in E. coli and purification of Phn-polypeptides. PhnG, PhnH, PhnI, PhnJ, and PhnK copurify as a protein complex by ion-exchange, size-exclusion, and affinity chromatography. The five polypeptides also comigrate in native-PAGE. Cross-linking of the purified protein complex reveals a close proximity of PhnG, PhnI, PhnJ, and PhnK, as these subunits disappear concomitant with the formation of large cross-linked protein complexes. Two molecular forms are identified, a major form of molecular mass of approximately 260 kDa, a minor form of approximately 640 kDa. The stoichiometry of the protein complex is suggested to be PhnG4H2I2J2K. Deletion of individual phn genes reveals that a strain harboring plasmid-borne phnGHIJ produces a protein complex consisting of PhnG, PhnH, PhnI, and PhnJ, whereas a strain harboring plasmid-borne phnGIJK produces a protein complex consisting of PhnG and PhnI. We conclude that phnGHIJK specify a soluble multisubunit protein complex essential for organophosphonate utilization.

Utilization of Glyphosate as Phosphate Source: Biochemistry and Genetics of Bacterial Carbon-Phosphorus Lyase

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.

Expression of nodule-specific uricase in soybean callus tissue is regulated by oxygen.

In soybean root nodules the enzyme uricase is expressed concomitantly with nodule development. The initial expression of this protein does not depend on active nitrogen fixation, as demonstrated by analysis of uricase activity in effective and ineffective root nodules. However, the maximal level of uricase activity is determined by the infecting Rhizobium japonicum strain. Sterile root cultures and callus tissue, devoid of the microsymbiont, were incubated at varying oxygen concentrations and analyzed for uricase activity. The specific activity of uricase was increased by lowering the oxygen concentration, with the highest activity obtained around 4−5% oxygen. The increase in uricase activity was due to increased uricase synthesis, as demonstrated by in vivo labelling of callus culture followed by immunoprecipitation with antibodies raised against highly purified nodule uricase.

Oxygen regulation of uricase and sucrose synthase synthesis in soybean callus tissue is exerted at the mRNA level

The effect of lowering oxygen concentration on the expression of nodulin genes in soybean callus tissue devoid of the microsymbiont has been examined. Poly(A)+ RNA was isolated from tissue cultivated in 4% oxygen and in normal atmosphere.Quantitative mRNA hybridization experiments using nodule-specific uricase (Nodulin-35) and sucrose synthase (Nodulin-100) cDNA probes confirmed that the synthesis of the uricase and sucrose synthase is controlled by oxygen at the mRNA level.The steady-state levels of uricase and sucrose synthase mRNA increased significantly (5–6- and 4-fold respectively) when the callus tissue was incubated at reduced oxygen concentration. Concomitant with the increase in mRNA level a 6-fold increase in specific activity of sucrose synthase was observed.Two messengers representing poly-ubiquitin precursors also responded to lowering the oxygen concentration. The increase was about 5-fold at 4% oxygen. No expression at atmospheric oxygen or in response to low oxygen was observed when using cDNA probes for other nodulin genes such as leghemoglobin c3, nodulin-22 and nodulin-44.

Structural insights into the bacterial carbon-phosphorus lyase machinery

Phosphorus 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 phosphonate compounds, which require specialized enzymatic machinery to break the stable carbon–phosphorus (C–P) bond. Despite its importance, the details of how this machinery catabolizes phosphonates remain unknown. Here we determine the crystal structure of the 240-kilodalton Escherichia coli C–P lyase core complex (PhnG–PhnH–PhnI–PhnJ; PhnGHIJ), and show that it is a two-fold symmetric hetero-octamer comprising an intertwined network of subunits with unexpected self-homologies. It contains two potential active sites that probably couple 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 a conserved insertion domain of PhnJ. Our results provide a structural basis for understanding microbial phosphonate breakdown.

Proteome reference maps of the Lotus japonicus nodule and root.

Legume symbiosis with rhizobia results in the formation of a specialized organ, the root nodule, where atmospheric dinitrogen is reduced to ammonia. In Lotus japonicus (Lotus), several genes involved in nodule development or nodule function have been defined using biochemistry, genetic approaches, and high‐throughput transcriptomics. We have employed proteomics to further understand nodule development. Two developmental stages representing nodules prior to nitrogen fixation (white) and mature nitrogen fixing nodules (red) were compared with roots. In addition, the proteome of a spontaneous nodule formation mutant (snf1) was determined. From nodules and roots, 780 and 790 protein spots from 2D gels were identified and approximately 45% of the corresponding unique gene accessions were common. Including a previous proteomics set from Lotus pod and seed, the common gene accessions were decreased to 7%. Interestingly, an indication of more pronounced PTMs in nodules than in roots was determined. Between the two nodule developmental stages, higher levels of pathogen‐related 10 proteins, HSPs, and proteins involved in redox processes were found in white nodules, suggesting a higher stress level at this developmental stage. In contrast, protein spots corresponding to nodulins such as leghemoglobin, asparagine synthetase, sucrose synthase, and glutamine synthetase were prevalent in red nodules. The distinct biochemical state of nodules was further highlighted by the conspicuous presence of several nitrilases, ascorbate metabolic enzymes, and putative rhizobial effectors.

Proteome reference maps of the Lotus japonicus nodule and root

Legume symbiosis with rhizobia results in the formation of a specialized organ, the root nodule, where atmospheric dinitrogen is reduced to ammonia. In Lotus japonicus (Lotus), several genes involved in nodule development or nodule function have been defined using biochemistry, genetic approaches, and high‐throughput transcriptomics. We have employed proteomics to further understand nodule development. Two developmental stages representing nodules prior to nitrogen fixation (white) and mature nitrogen fixing nodules (red) were compared with roots. In addition, the proteome of a spontaneous nodule formation mutant (snf1) was determined. From nodules and roots, 780 and 790 protein spots from 2D gels were identified and approximately 45% of the corresponding unique gene accessions were common. Including a previous proteomics set from Lotus pod and seed, the common gene accessions were decreased to 7%. Interestingly, an indication of more pronounced PTMs in nodules than in roots was determined. Between the two nodule developmental stages, higher levels of pathogen‐related 10 proteins, HSPs, and proteins involved in redox processes were found in white nodules, suggesting a higher stress level at this developmental stage. In contrast, protein spots corresponding to nodulins such as leghemoglobin, asparagine synthetase, sucrose synthase, and glutamine synthetase were prevalent in red nodules. The distinct biochemical state of nodules was further highlighted by the conspicuous presence of several nitrilases, ascorbate metabolic enzymes, and putative rhizobial effectors.

The Cockayne syndrome group B protein is a functional dimer

Cockayne syndrome (CS) is a rare inherited human genetic disorder characterized by developmental abnormalities, UV sensitivity, and premature aging. The CS group B (CSB) protein belongs to the SNF2‐family of DNA‐dependent ATPases and is implicated in transcription elongation, transcription coupled repair, and base excision repair. It is a DNA stimulated ATPase and remodels chromatin in vitro. We demonstrate for the first time that full‐length CSB positively cooperates in ATP hydrolysis as a function of protein concentration. We have investigated the quaternary structure of CSB using a combination of protein–protein complex trapping experiments and gel filtration, and found that CSB forms a dimer in solution. Chromatography studies revealed that enzymatically active CSB has an apparent molecular mass of approximately 360 kDa, consistent with dimerization of CSB. Importantly, in vivo protein cross‐linking showed the presence of the CSB dimer in the nucleus of HeLa cells. We further show that dimerization occurs through the central ATPase domain of the protein. These results have implications for the mechanism of action of CSB, and suggest that other SNF2‐family members might also function as dimers.

A Proton Wire and Water Channel Revealed in the Crystal Structure of Isatin Hydrolase

Background: Isatin hydrolase is involved in bacterial indole-3-acetic acid degradation and belongs to a structurally uncharacterized family. Results: We determined the first crystal structure of a functionally characterized metal dependent hydrolase of this overall fold. Conclusion: The new hydrolytic fold reveals a transient water wire that allow proton release. Significance: Proton transfer is a fundamental process important in the understanding of enzymes and membrane transporters. The high resolution crystal structures of isatin hydrolase from Labrenzia aggregata in the apo and the product state are described. These are the first structures of a functionally characterized metal-dependent hydrolase of this fold. Isatin hydrolase converts isatin to isatinate and belongs to a novel family of metalloenzymes that include the bacterial kynurenine formamidase. The product state, mimicked by bound thioisatinate, reveals a water molecule that bridges the thioisatinate to a proton wire in an adjacent water channel and thus allows the proton released by the reaction to escape only when the product is formed. The functional proton wire present in isatin hydrolase isoform b represents a unique catalytic feature common to all hydrolases is here trapped and visualized for the first time. The local molecular environment required to coordinate thioisatinate allows stronger and more confident identification of orthologous genes encoding isatin hydrolases within the prokaryotic kingdom. The isatin hydrolase orthologues found in human gut bacteria raise the question as to whether the indole-3-acetic acid degradation pathway is present in human gut flora.