Identification and cloning of nodulation genes from the wide host range Rhizobium strain MPIK3030

Summary A 70 kbp segment of the megaplasmid from a broad host range Rhizobium strain (MPIK3030) was mapped with the aid of cosmid clones made in the vector pJB8. A 7.9 kbp EcoRI fragment from this region, 55 kbp away from the nif gene cluster, was shown to hybridize to the common nod genes from R. meliloti. Using several R. meliloti nod probes it was possible to delimit an 830 bp region as being the center of greatest homology. Sequence data from two sections of this region gave a nucleotide homology of 73.7% to the nodC gene of R. meliloti. Using Tn5 mutagenesis a clone was isolated carrying Tn5 in the highly homologous region. When tested on Macroptilium atropurpureum, this MPIK3030 derivative was shown to have a Nod^– phenotype. When the wild-type allele was reintroduced into the Tn5 mutant, nodulation was restored. Interspecies complementation also showed that both R. meliloti and Rhizobium sp. MPIK3030 nod regions were able to restore nodulation to Tn5-induced nodC mutants from either strain.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

The crystal structure of archaeal nascent polypeptide-associated complex (NAC) reveals a unique fold and the presence of a ubiquitin-associated domain

Nascent polypeptide-associated complex (NAC) was identified in eukaryotes as the first cytosolic factor that contacts the nascent polypeptide chain emerging from the ribosome. NAC is highly conserved from yeast to humans. Mutations in NAC cause severe embryonically lethal phenotypes in mice, Drosophila, and Caenorhabditis elegans. NAC was suggested to protect the nascent chain from inappropriate early interactions with cytosolic factors. Eukaryotic NAC is a heterodimer with two subunits sharing substantial homology with each other. All sequenced archaebacterial genomes exhibit only one gene homologous to the NAC subunits. Here we present the first archaebacterial NAC homolog. It forms a homodimer, and as eukaryotic NAC it is associated with ribosomes and contacts the emerging nascent chain on the ribosome. We present the first crystal structure of a NAC protein revealing two structural features: (i) a novel unique protein fold that mediates dimerization of the complex, and (ii) a ubiquitin-associated domain that suggests a yet unidentified role for NAC in the cellular protein quality control system via the ubiquitination pathway. Based on the presented structure we propose a model for the eukaryotic heterodimeric NAC domain.     

Searching for intermediates in the carbon skeleton rearrangement of 2-methyleneglutarate to (R)-3-methylitaconate catalyzed by coenzyme B12-dependent 2-methyleneglutarate mutase from Eubacterium barkeri

Coenzyme B(12)-dependent 2-methyleneglutarate mutase from the strict anaerobe Eubacterium barkeri catalyzes the equilibration of 2-methyleneglutarate with (R)-3-methylitaconate. Proteins with mutations in the highly conserved coenzyme binding-motif DXH(X)(2)G(X)(41)GG (D483N and H485Q) exhibited decreased substrate turnover by 2000-fold and >4000-fold, respectively. These findings are consistent with the notion of H485 hydrogen-bonded to D483 being the lower axial ligand of adenosylcobalamin in 2-methyleneglutarate mutase. (E)- and (Z)-2-methylpent-2-enedioate and all four stereoisomers of 1-methylcyclopropane-1,2-dicarboxylate were synthesized and tested, along with acrylate, with respect to their inhibitory potential. Acrylate and the 2-methylpent-2-enedioates were noninhibitory. Among the 1-methylcyclopropane-1,2-dicarboxylates only the (1R,2R)-isomer displayed weak inhibition (noncompetitive, K(i) = 13 mM). Short incubation (5 min) of 2-methyleneglutarate mutase with 2-methyleneglutarate under anaerobic conditions generated an electron paramagnetic resonance (EPR) signal (g(xy) approximately 2.1; g(z) approximately 2.0), which by analogy with the findings on glutamate mutase from Clostridium cochlearium [Biochemistry, 1998, 37, 4105-4113] was assigned to cob(II)alamin coupled to a carbon-centered radical. At longer incubation times (>1 h), inactivation of the mutase occurred concomitant with the formation of oxygen-insensitive cob(II)alamin (g(xy) approximately 2.25; g(z) approximately 2.0). In order to identify the carbon-centered radical, various (13)C- and one (2)H-labeled substrate/product molecules were synthesized. Broadening (0.5 mT) of the EPR signal around g = 2.1 was observed only when C2 and/or C4 of 2-methyleneglutarate was labeled. No effect on the EPR signals was seen when [5'-(13)C]adenosylcobalamin was used as coenzyme. The inhibition and EPR data are discussed in the context of the addition-elimination and fragmentation-recombination mechanisms proposed for 2-methyleneglutarate mutase.     

A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo

Riboswitches are newly discovered regulatory elements which control a wide set of basic metabolic pathways. They consist solely of RNA, sense their ligand in a preformed binding pocket and perform a conformational switch in response to ligand binding resulting in altered gene expression. We have utilized the enormous potential of RNA for molecular sensing and conformational changes to develop novel molecular switches with predetermined structural transitions in response to the binding of a small molecule. To validate these in vivo, we exploit the distance-dependent inhibitory potential of secondary structure elements placed close to the bacterial ribosome binding site. We created a translational control element by combining the theophylline aptamer with a helical communication module for which a ligand-dependent one-nucleotide slipping mechanism had been proposed. This structural element was inserted at a position just interfering with translation in the non ligand-bound form. Addition of the ligand then shifts the inhibitory element to a distance which permits efficient translation. We present here a novel regulatory mechanism in the first rationally designed, in vivo active RNA switch. Its use of a slippage mechanism to control gene expression makes it different from natural riboswitches which are based on sequestration or antitermination.     

Analysis of the cytosolic proteome of Halobacterium salinarum and its implication for genome annotation

The halophilic archaeon Halobacterium salinarum (strain R1, DSM 671) contains 2784 protein-coding genes as derived from the genome sequence. The cytosolic proteome containing 2042 proteins was separated by two-dimensional gel electrophoresis (2-DE) and systematically analyzed by a semi-automatic procedure. A reference map was established taking into account the narrow isoelectric point (pI) distribution of halophilic proteins between 3.5 and 5.5. Proteins were separated on overlapping gels covering the essential areas of pI and molecular weight. Every silver-stained spot was analyzed resulting in 661 identified proteins out of about 1800 different protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting (PMF). There were 94 proteins that were found in multiple spots, indicating post-translational modification. An additional 141 soluble proteins were identified on 2-D gels not corresponding to the reference map. Thus about 40% of the cytosolic proteome was identified. In addition to the 2784 protein-coding genes, the H. salinarum genome contains more than 6000 spurious open reading frames longer than 100 codons. Proteomic information permitted an improvement in genome annotation by validating and correcting gene assignments. The correlation between theoretical pI and gel position is exceedingly good and was used as a tool to improve start codon assignments. The fraction of identified chromosomal proteins was much higher than that of those encoded on the plasmids. In combination with analysis of the GC content this observation permitted an unambiguous identification of an episomal insert of 60 kbp ("AT-rich island") in the chromosome, as well as a 70 kbp region from the chromosome that has integrated into one of the megaplasmids and carries a series of essential genes. About 63% of the chromosomally encoded proteins larger than 25 kDa were identified, proving the efficacy of 2-DE MALDI-TOF MS PMF technology. The analysis of the integral membrane proteome by tandem mass spectrometric techniques added another 141 identified proteins not identified by the 2-DE approach (see following paper).     

The biotin-dependent sodium ion pump glutaconyl-CoA decarboxylase from Fusobacterium nucleatum (subsp. nucleatum)

Membrane preparations of Fusobacterium nucleatum grown on glutamate contain glutaconyl-CoA decarboxylase at a high specific activity (13.8 nkat/mg protein). The enzyme was solubilized with 2% Triton X-100 in 0.5M NaCl and purified 63-fold to a specific activity of 870 nkat/mg by affinity chromatography on monomeric avidin-Sepharose. The activity of the decarboxylase was strictly dependent on Na⁺ (K _m=3 mM) and was stimulated up to 3-fold by phospholipids. The glutaconyl-CoA decarboxylases from the gram-positive bacteria Acidaminococcus fermentans and Clostridium symbiosum have a lower apparent K _m for Na⁺ (1 mM) and were not stimulated by phospholipids. In addition only the fusobacterial decarboxylase required sodium ion for stability and was inactivated by potassium ion. By incorporation of this purified enzyme into phospholipids an electrogenic sodium ion pump was reconstituted. The enzyme consists of four subunits, (m=65 kDa), (33 kDa), (19 kDa), and (16 kDa) with the functions of a carboxy transferase (), a carboxy lyase ( and probably ) and a biotin carrier (). The subunits are very similar to those of the glutaconyl-CoA decarboxylases from the gram-positive bacteria. With an antiserum directed against the decarboxylase from A. fermentans the - and the biotin containing subunits of the three decarboxylases and that from Peptostreptoccus asaccharolyticus could be detected on Western blots.     

Determination of physical membrane properties of plant cell protoplasts via the electrofusion technique: prediction of optimal fusion yields and protoplast viability

By variation of physical parameters (field strength, pulse duration) which result in electrofusion and electroporation, properties of the plasma membrane of different types of plant cell protoplasts were analyzed. The lower threshold for that field pulse intensity at which membrane breakdown occurred (recorded as fusion event) depended on pulse duration, protoplast size, and protoplast type (tobacco, oat; vacuolated, evacuolated). This fusion characteristic of plant protoplasts can also be taken as a measure of the charging process of the membrane and allows thus a non-invasive determination of the time constant and the specific membrane capacitance. Although the fusion yield was comparable at pulse duration/field strength couples of, e.g., 10 s/1.5 kV*cm^–1 and 200 s/0.5 kV*cm^–1, hybrid viability was not. Rates of cell wall regeneration and cell division of tobacco mesophyll protoplasts were not affected but may have been increased at short pulse duration/high field strength. Plating efficiency, in contrast, was significantly decreased with longer pulse duration at low field strengths.     

Anaerobic metabolism in aquatic insect larvae: studies onChironomus thummi andCulex pipiens

Summary The metabolic effect of hypoxia and anoxia on the larvae ofChironomus thummi andCulex pipiens was investigated. InC. thummi anoxia resulted in a characteristic decrease of ATP and P-arginine concentrations and in an accumulation of alanine and lactate within 60 minutes. These changes continued during prolonged incubation but at lower rates. Ethanol, the major product during long-term anoxia, was largely excreted into the ambient water.A significant accumulation of these metabolites occurred only at a of 7 Torr. However, the proportion of anaerobic energy production even at this low amounted to less than 5% of the total energy consumption measured during experimental anoxia. Thus the chironomid larvae exhibited a remarkable capacity for utilizing very low levels of oxygen to maintain an aerobic metabolism. Complete anaerobiosis was observed only under anoxic conditions.Recovery from prior anoxia began with the reestablishment of normal ATP, P-arginine and succinate concentrations, whereas removal of the accumulated alanine and lactate and replenishment of the normally high level of malate required several hours. Culex larvae were shown to have a very low anaerobic capacity and a high rate of lactate accumulation.The significance of the results is discussed with particular emphasis on comparative aspects.     

Selection of an Asporogenous Strain of Clostridium acetobutylicum in Continuous Culture Under Phosphate Limitation

Based on the observation that cells of Clostridium acetobutylicum unable to store granulose do not initiate sporulation, a staining procedure was developed for the detection of asporogenous mutants. By application of this procedure it was shown that an asporogenous strain of C. acetobutylicum was selected in continuous culture under phosphate limitation.     

Variability in the synthesis of cytochromes f and b-563 during the cell cycle of Euglena gracilis

The study of the successive formation of the photosynthetic apparatus in Euglena gracilis (Brandt, P. and Von Kessel, B. (1983) Plant Physiol. 72, 616–619) was extended to the determination of the stage-specific synthesis of cytochrome $\text{b}\text{f}$ complex during the cell cycle of this alga. Most of the cytochrome f (33 kDa) has properties of an intrinsic membrane protein, but part of it is soluble. Cytochrome b-563 (18 kDa) is only intrinsic. The intensity of binding the intrinsic cytochromes in the thylakoids depends on the developmental stage of the organism. The light-independent synthesis of cytochrome f takes place prior to the assembly of the chlorophyll-protein complex I (CP I). Immediately after this assembly of CP I, cytochrome b-563 is synthesized in the light. Hence, the ratio cytochrome b-563/cytochrome f changes during the cell cycle of E. gracilis. The physiological implication of presumably non-complexed cytochrome f and of complex-bound cytochromes f and b-563 on the stage-specific efficiency of photosynthesis of E. gracilis is discussed.