In vitro studies with purified components reveal signal recognition particle (SRP) and SecA/SecB as constituents of two independent protein-targeting pathways of Escherichia coli.

The molecular requirements for the translocation of secretory proteins across, and the integration of membrane proteins into, the plasma membrane of Escherichia coli were compared. This was achieved in a novel cell-free system from E. coli which, by extensive subfractionation, was simultaneously rendered deficient in SecA/SecB and the signal recognition particle (SRP) components, Ffh (P48), 4. 5S RNA, and FtsY. The integration of two membrane proteins into inside-out plasma membrane vesicles of E. coli required all three SRP components and could not be driven by SecA, SecB, and DeltamicroH+. In contrast, these were the only components required for the translocation of secretory proteins into membrane vesicles, a process in which the SRP components were completely inactive. Our results, while confirming previous in vivo studies, provide the first in vitro evidence for the dependence of the integration of polytopic inner membrane proteins on SRP in E. coli. Furthermore, they suggest that SRP and SecA/SecB have different substrate specificities resulting in two separate targeting mechanisms for membrane and secretory proteins in E. coli. Both targeting pathways intersect at the translocation pore because they are equally affected by a blocked translocation channel.

Oxazolidinones: a novel class of antibiotics.

Oxazolidinones are a novel class of synthetic antimicrobial agents which have now entered phase III clinical trials. The most promising feature of these compounds is their oral activity against multidrug-resistant Gram-positive bacteria which have created tremendous therapeutic problems in recent years. In addition, development of resistance in vitro has so far remained below detectable levels. Different from many antibacterial agents used in the treatment of human infections, oxazolidinones do not block bacterial protein synthesis at the level of polypeptide chain elongation but rather seem to interfere with initiation of translation. Both binding of formylmethionine-transfer RNA to initiation complexes as well as release of formylmethioninepuromycin from initiation complexes have been reported to be targets for oxazolidinones. The major binding sites of oxazolidinones are the large (50S) ribosomal subunits.

On the target of a novel class of antibiotics, oxazolidinones, active against multidrug-resistant Gram-positive bacteria.

Oxazolidinones are a promising new class of synthetic antibiotics active against multidrug-resistant Gram-positive bacteria. To elucidate their mode of action, the effect of DuP 721 on individual steps of protein translation was studied. The drug does not interfere with translation initiation at the stage of mRNA binding or formation of 30S pre-initiation complexes. However, it inhibits the puromycin-mediated release of [35S]formyl-methionine from 70S initiation complexes in a dose-dependent manner. Inhibition involves binding of the oxazolidinone to the large ribosomal subunit and is twice as high with 50S subunits from Gram-positive as with those from Gram-negative bacteria.

Comparative characterization of SecA from the alpha-subclass purple bacterium Rhodobacter capsulatus and Escherichia coli reveals differences in membrane and precursor specificity.

We have cloned the secA gene of the alpha-subclass purple bacterium Rhodobacter capsulatus, a close relative to the mitochondrial ancestor, and purified the protein after expression in Escherichia coli. R. capsulatus SecA contains 904 amino acids with 53% identity to E. coli and 54% identity to Caulobacter crescentus SecA. In contrast to the nearly equal partitioning of E. coli SecA between the cytosol and plasma membrane, R. capsulatus SecA is recovered predominantly from the membrane fraction. A SecA-deficient, cell-free synthesis-translocation system prepared from R. capsulatus is used to demonstrate translocation activity of the purified R. capsulatus SecA. This translocation activity is then compared to that of the E. coli counterpart by using various precursor proteins and inside-out membrane vesicles prepared from both bacteria. We find a preference of the R. capsulatus SecA for the homologous membrane vesicles whereas E. coli SecA is active with either type of membrane. Furthermore, the two SecA proteins clearly select between distinct precursor proteins. In addition, we show here for the first time that a bacterial c-type cytochrome utilizes the canonical, Sec-dependent export pathway.

A cell-free protein translocation system prepared entirely from a gram-positive organism.

A cell-free protein translocation system derived exclusively from a Gram-positive bacterium is described here for the first time. Highly efficient in vitro synthesis of plasmid encoded preprolipase of Staphylococcus hyicus is accomplished by coupled transcription/translation using either a cytosolic extract of S. carnosus alone or in combination with T7-RNA-polymerase. Addition of inside-out cytoplasmic membrane vesicles of S. carnosus leads to the partial conversion (processing) of preprolipase to prolipase. In addition, as shown in a protease protection assay, a significant part of preprolipase plus prolipase is translocated in vitro into the lumen of the vesicles. Translocation of preprolipase into the membrane vesicles requires the proton-motive force and the S. carnosus SecA protein.

Lysine 106 of the putative catalytic ATP-binding site of the Bacillus subtilis SecA protein is required for functional complementation of Escherichia coli secA mutants in vivo.

The SecA protein is a major component of the cellular machinery that mediates the translocation of proteins across the Escherichia coli plasma membrane. The secA gene from Bacillus subtilis was cloned and expressed in E. coli under the control of the lac or trc promoter. The temperature-sensitive growth and secretion defects of various E. coli secA mutants were complemented by the B. subtilis SecA protein, provided the protein was expressed at moderate levels. Under overproduction conditions, no complementation was observed. One of the main features of the SecA protein is the translocation ATPase activity which, together with the protonmotive force, drives the movement of proteins across the plasma membrane. A putative ATP-binding motif can be identified in the SecA protein resembling the consensus Walker A type motif. Replacement of a lysine residue at position 106, which corresponds to an invariable amino acid residue, in the consensus motif by asparagine (K106N) resulted in the loss of the ability of the B. subtilis SecA protein to complement the growth and secretion defects of E. coli secA mutants. In addition, the presence of the K106N SecA protein interfered with protein translocation, most likely at an ATP-requiring step. We conclude that lysine 106 is part of the catalytic ATP-binding site of the B. subtilis SecA protein, which is required for protein translocation in vivo.

Degradation of Extracellular beta-(1,3)(1,6)-d-Glucan by Botrytis cinerea.

During growth on glucose, Botrytis cinerea produced extracellular beta-(1,3)(1,6)-d-glucan (cinerean), which formed an adhering capsule and slime. After glucose was exhausted from the medium, cinereanase activity increased from <0.4 to 30 U/liter, effecting a striking loss in the viscosity of the culture. Cinerean was cleaved into glucose and gentiobiose. Gentiobiose was then hydrolyzed to glucose. While cinereanase activity was strongest in the culture supernatant, gentiobiase activity was located mainly in the cell wall fraction. The addition of extra glucose or cycloheximide prevented the cinerean degradation caused by an effect on cinereanase formation. Cinerean degradation was accompanied by microconidiation and sclerotium formation. B. cinerea was found to grow on cinerean with the latter as its single carbon and energy source. In this case, cinerean degradation occurred during hyphal growth, and no microconidiation or sclerotium formation was observed. Growth experiments with various carbon sources indicated that cinerean had a positive effect on the formation of cinerean-degrading enzymes.

Determination of intracellular pyridine nucleotide levels by bioluminescence using anaerobic bacteria as a model.

An assay for the determination of NAD(P)+ and NAD(P)H in extracts from the obligate anaerobe bacterium Thermoanaerobacter finnii is developed and the strategy for this development is described. This assay performed with constant FMN reductase (EC 1.6.8.1) and luciferase (EC 1.14.14.3) concentrations has been shown to detect as low as 1 pmol pyridine nucleotide. With this assay recovery, efficiency of extraction, reliability, and detection limit of the procedures were determined. To our knowledge this is the first bioluminometric assay for the determination of pyridine nucleotide levels in bacterial extracts.

Glucose metabolism in Xanthomonas campestris and influence of methionine on the carbon flow.

The glucose flow in Xanthomonas campestris was investigated with radio-labelled glucose and by enzymological studies. Only 7% of the radioactivity was incorporated into the cell material, but 41% was oxidized to carbon dioxide and 28% transformed to xanthan. Up to 16% of cell dry weight consisted of the polysaccharide glycogen. In the presence of 2.7 mM methionine, which is an inhibitor of xanthan formation, increased carbon dioxide formation (51%) occurred. This increase was in accordance with a twofold increase in the NAD-dependent isocitrate dehydrogenase activity. The other carbon dioxide liberating enzyme, 6-P-gluconate dehydrogenase, was not influenced by methionine, but its occurrence indicates the presence of an active pentose phosphate pathway in X. campestris. Among the other enzymes detected in X. campestris was glucose dehydrogenase. The presence of this enzyme together with hexokinase indicates the operation of two different glucose metabolizing steps: one oxidative, the other phosphorylative. Only the latter directly provides phosphorylated glucose as a precursor for the activated sugars required for xanthan synthesis.

The effect of sulfite on the yeast Saccharomyces cerevisiae.

After a short period of tolerance, living cells of Saccharomyces cerevisiae were irreversibly damaged by low concentrations of sulfite. The length of the period of tolerance and the rate of the damaging effect depended on the concentration on sulfite, pH-value, temperature, the physiological state of the cells, and incubation time. Inhibitors of protein synthesis and mitochondrial ATP synthesis did not alter the deleterious effect of sulfite on living cells. Furthermore, cell damage leading to inhibition of colony formation occured under aerobic as well as under anaerobic conditions. Prior to cell inactivation sulfite induced the formation of respiratory deficient cells. The active agent was shown to be SO2.

Rapid decrease of ATP content in intact cells of Saccharomyces cerevisiae after incubation with low concentrations of sulfite.

Sulfite, at concentrations above 1 mM and at a pH below 4, caused cell death in Saccharomyces cerevisiae X2180 as measured by the colony-forming capacity. A rapid decrease in the ATP content was observed prior to cellular death. The depletion of ATP was reversible and the lethal effect could be prevented if the cells were exposed to sulfite for periods of less than 1 h. Extent and rate of ATP depletion were dependent on time, pH value, temperature and sulfite concentrations.

D-glucose-6-phosphate dehydrogenase (Entner-Doudoroff enzyme) from Pseudomonas fluorescens. Purification, properties and regulation.

1. The existence of two different D-glucose-6-phosphate dehydrogenases in Pseudomonas fluorescens has been demonstrated. Based on their different specificity and their different metabolic regulation one enzyme is appointed to the Entner-Doudoroff pathway and the other to the hexose monophosphate pathway. 2. A procedure is described for the isolation of that D-glucose-6-phosphate dehydrogenase which forms part of the Entner-Doudoroff pathway (Entner-Doudoroff enzyme). A 950-fold purification was achieved with an overall yield of 44%. The final preparation, having a specific activity of about 300 mumol NADH formed per min per mg protein, was shown to be homogeneous. 3. The molecular weight of the Entner-Doudoroff enzyme has been determined to be 220000 by gel permeation chromatography, and that of the other enzyme (Zwischenferment) has been shown to be 265000. 4. The pI of the Entner-Doudoroff enzyme has been shown to be 5.24 and that of the Zwischenferment 4.27. The Entner-Doudoroff enzyme is stable in the range of pH 6 to 10.5 and shows its maximal activity at pH 8.9. 5. The Entner-Doudoroff enzyme showed specificity for NAD+ as well as for NADP+ and exhibited homotropic effects for D-glucose 6-phosphate. It is inhibited by ATP which acts as a negative allosteric effector. Other nucleoside triphosphates as well as ADP are also inhibitory. 6. The enzyme catalyzes the transfer of the axial hydrogen at carbon-1 of beta-D-glucopyranose 6-phosphate to the si face of carbon-4 of the nicotinamide ring and must be classified as B-side stereospecific dehydrogenase.