Role of the two-component leader sequence and mature amino acid sequences in extracellular export of endoglucanase EGL from Pseudomonas solanacearum.

The egl gene of Pseudomonas solanacearum encodes a 43-kDa extracellular endoglucanase (mEGL) involved in wilt disease caused by this phytopathogen. Egl is initially translated with a 45-residue, two-part leader sequence. The first 19 residues are apparently removed by signal peptidase II during export of Egl across the inner membrane (IM); the remaining residues of the leader sequence (modified with palmitate) are removed during export across the outer membrane (OM). Localization of Egl-PhoA fusion proteins showed that the first 26 residues of the Egl leader sequence are required and sufficient to direct lipid modification, processing, and export of Egl or PhoA across the IM but not the OM. Fusions of the complete 45-residue leader sequence or of the leader and increasing portions of mEgl sequences to PhoA did not cause its export across the OM. In-frame deletion of portions of mEGL-coding sequences blocked export of the truncated polypeptides across the OM without affecting export across the IM. These results indicate that the first part of the leader sequence functions independently to direct export of Egl across the IM while the second part and sequences and structures in mEGL are involved in export across the OM. Computer analysis of the mEgl amino acid sequence obtained from its nucleotide sequence identified a region of mEGL similar in amino acid sequence to regions in other prokaryotic endoglucanases.     

Techniques in plant molecular biology--progress and problems

Progress in plant molecular biology has been dependent on efficient methods of introducing foreign DNA into plant cells. Gene transfer into plant cells can be achieved by either direct uptake of DNA or the natural process of gene transfer carried out by the soil bacterium Agrobacterium. Versatile gene-transfer vectors have been developed for use with Agrobacterium and more recently vectors based on the genomes of plant viruses have become available. Using this technology the expression of foreign DNA, the functional analysis of plant DNA sequences, the investigation of the mechanism of viral DNA replication and cell to cell spread, as well as the study of transposition, can be carried out. In addition, the versatility of the gene-transfer vectors is such that they may be used to isolate genes not amenable to isolation using conventional protocols. This review concentrates on these aspects of plant molecular biology and discusses the limitations of the experimental systems that are currently available.     

Broad-host-range cloning vectors derived from the W-plasmid Sa

Four nonconjugative broad-host-range cloning vectors were derived from the W-plasmid Sa. They are small (M_r 5.6?7.2 × 10⁶), carry several drug-resistance markers, and allow constructing and screening for recombinant plasmids generated by the restriction enzymes EcoRI, PstI, BglII, HindIII, BamHI and SalI,     

Sensitive and Accurate Methodology for Measuring the Kinetics of Concentration-Dependent Hydrocarbon Metabolism Rates in Seawater by Microbial Communities

A method having sufficient sensitivity to resolve the kinetic constants for dissolved nonpolar substrate metabolism, together with the related rate constants in natural waters, is presented. The method is based on the rate of ¹⁴CO₂ recovery from radioactive dissolved substrate. Sensitivity is enhanced by using large seawater volumes, high-specific-activity isotopes, and by reducing background radioactivity. Before use, commercial isotopes are purified by mild alkaline hydrolysis followed by sublimation from base to remove ¹⁴CO₂ as well as interfering polar ¹⁴C-substrates. During sample analysis, chilled Tenax resin is used to remove volatile ¹⁴C-substrate from the nitrogen stream containing ¹⁴CO₂ recovered from substrate oxidation. Chromatographic evidence of purity, shown to be insufficient, is augmented by kinetic data from toluene utilization by mixed cultures and by rates in induced versus noninduced pure cultures. Accuracy is enhanced by using short (<10 h) incubation times and small hydrocarbon concentrations so that the metabolism rates in unamended natural water systems can be evaluated. Toluene metabolism rates in seawater as low as 1 pg/liter per h and at concentrations as low as 20 ng/liter have been determined.     

Reconstitution of acetylcholine receptor from Torpedo californica with highly purified phospholipids: Effect of α-tocopherol,phylloquinone, and other terpenoid quinones

Acetylcholine receptor from $\text{Torpedo californica}$ can be incorporated by the cholate dialysis procedure into liposomes prepared with crude soybean phospholipids (asolectin). Vesicles reconstituted with asolectin depleted of neutral lipids or with a mixture of pure phospholipids, are less active in catalyzing carbamylcholine-sensitive Na⁺ flux. Inclusion of α-tocopherol or certain quinones such as coenzyme Q₁₀ or vitamin K₁ during reconstitution yields vesicles with carbamylcholine-sensitive Na⁺ flux which, under optimal conditions, was considerably higher than that observed with vesicles reconstituted with crude phospholipid mixtures.