A hybrid toxin from bacteriophage f1 attachment protein and colicin E3 has altered cell receptor specificity.

A hybrid protein was constructed in vitro which consists of the first 372 amino acids of the attachment (gene III) protein of filamentous bacteriophage f1 fused, in frame, to the carboxy-terminal catalytic domain of colicin E3. The hybrid toxin killed cells that had the F-pilus receptor for phage f1 but not F- cells. The activity of the hybrid protein was not dependent upon the presence of the colicin E3 receptor, BtuB protein. The killing activity was colicin E3 specific, since F+ cells expressing the colicin E3 immunity gene were not killed. Entry of the hybrid toxin was also shown to depend on the products of tolA, tolQ, and tolR which are required both for phage f1 infection and for entry of E colicins. TolB protein, which is required for killing by colicin E3, but not for infection by phage f1, was also found to be necessary for the killing activity of the hybrid toxin. The gene III protein-colicin E3 hybrid was released from producing cells into the culture medium, although the colicin E3 lysis protein was not present in those cells. The secretion was shown to depend on the 18-amino-acid-long gene III protein signal sequence. Deletion of amino acids 3 to 18 of the gene III moiety of the hybrid protein resulted in active toxin, which remained inside producing cells unless it was mechanically released.     

Cyclic AMP-induced conformational change of cyclic AMP receptor protein (CRP): intragenic suppressors of cyclic AMP-independent CRP mutations.

We isolated and characterized crp mutations in Escherichia coli that allow cyclic AMP (cAMP) receptor protein to function without cAMP. These mutants defined a region involved in the cAMP-induced allosteric change of cAMP receptor protein that is necessary for activation of the protein. Currently, we have isolated intragenic suppressors of the crp mutations. These crp (Sup) mutants require cAMP for activity. The crp (Sup) mutations map in regions which define new sites of changes involved in cAMP receptor protein activation. From these results, we suggest that to activate cAMP receptor protein cAMP brings about (i) a hinge reorientation to eject the DNA-binding F alpha-helices, (ii) proper alignment between the two subunits, and (iii) an adjustment between the position of the two domains. Cyclic GMP fails to effect the last step.     

Regulation of carbon flow in Selenomonas ruminantium grown in glucose-limited continuous culture.

We have applied a model that permits the estimation of the sensitivity of flux through branch point enzymes (D. C. LaPorte, K. Walsh, and D. E. Koshland, J. Biol. Chem. 259:14068-14075, 1984) in order to analyze the control of flux through the lactate-acetate branch point of Selenomonas ruminantium grown in glucose-limited continuous culture. At this branch point, pyruvate is the substrate of both the NAD-dependent L-(+)-lactate dehydrogenase (LDH) and the pyruvate:ferredoxin oxidoreductase (PFOR). The LDH was purified, and it exhibited positive cooperativity for the binding of pyruvate. The LDH had an [S].5 for pyruvate of 0.43 mM, a Hill coefficient of 2.4, and a K' equal to 0.13 mM. The PFOR, assayed in cell extracts, exhibited Michaelis-Menten kinetics for pyruvate, with a Km of 0.49 mM. Carbon flux through the LDH and the PFOR increased 80-fold and 3-fold, respectively, as the dilution rate was increased from 0.07 to 0.52 h-1 in glucose-limited continuous culture. There was nearly a twofold increase, from 6.5 to 11.2 mumol min-1 mg of protein-1 in the specific activity (i.e., maximum velocity) of the LDH at dilution rates of 0.11 and 0.52 h-1, respectively. A flux equation was used to calculate the intracellular concentration of pyruvate; a fourfold increase in pyruvate, from 0.023 to 0.093 mM, was thereby predicted as the dilution rate was increased from 0.07 to 0.52 h-1. When these calculated values of intracellular pyruvate concentration were inserted into the flux equation, the predicted values of flux through the LDH and the PFOR were found to match closely the flux actually measured in the chemostat-grown cells. Thus, the 80-fold increase in flux through the LDH was due to a twofold increase in the maximum velocity of the LDH and a fourfold increase in the intracellular pyruvate concentration. In addition, the flux through the LDH exhibited ultrasensitivity to changes in both the maximum velocity of the LDH and the intracellular concentration of pyruvate. The flux through the PFOR exhibited ultrasensitivity to changes in the maximum velocity of the LDH and hyperbolic sensitivity to changes in the intracellular concentration of pyruvate.     

Interference Competition among Coprophilous Fungi: Production of (+)-Isoepoxydon by Poronia punctata

(+)-Isoepoxydon has been established as the major causative agent of interference competition between Poronia punctata (NRRL 6457), a late fungal colonist of cattle dung, and two early-occurring dung colonists, Ascobolus furfuraceus (NRRL 6460) and Sordaria fimicola (NRRL 6459). This compound was isolated from ethyl acetate extracts of liquid cultures of P. punctata by silica gel chromatography and identified by mass spectrometry and proton and carbon nuclear magnetic resonance spectroscopy. The isolation process was guided by in vitro bioassays for antifungal activity against A. furfuraceus and S. fimicola. (+)-Isoepoxydon has been implicated as an intermediate in the biosynthesis of patulin, a mycotoxin produced by Penicillium spp., but no patulin could be detected in cultures of P. punctata.     

Isolation of a New Polysaccharide-Digesting Bacterium from a Salt Marsh

A new marine bacterium that digested a variety of storage and structural polysaccharides, including agar, was isolated. Strain 2-40 is a nonfermentative gram-negative, polarly flagellated rod that sometimes grew as a filamentous helix and secreted a melaninlike pigment. Its characteristics conform to those of no previously described species.     

Evaluation of four methods for enumeration of Vibrio parahaemolyticus.

Two membrane filter (MF) and two most-probable-number methods for enumerating Vibrio parahaemolyticus were compared. The MF methods used elevated-temperature incubations (41 and 42 degrees C) and were more specific than the most-probable-number methods (conducted at 35 degrees C). The MF method with a hydrophobic grid and a repair step was most effective.     

Development of a Mutant Strain of Bacillus polymyxa Showing Enhanced Production of 2,3-Butanediol

2,3-Butanediol is a feedstock chemical of potential industrial importance. It can serve as a monomer for many polymers of consumer interest that are currently supplied by the fossil fuel industry. Bacillus polymyxa can grow on inexpensive waste products of the food-processing industry and produce this glycol. This paper describes a mutant strain of B. polymyxa which displays constitutive production of catabolic α-acetolactate synthase, an enzyme in the 2,3-butanediol pathway which is normally produced only in the late log or stationary phase of growth. The mutant was obtained by treating the wild type with nitrosoguanidine and subjecting it to a penicillin counterselection procedure. One of the selected mutant strains produced four times as much of the glycol as the wild type and utilized approximately 25% of the energy source, compared with essentially complete utilization of the energy source by the wild type. Studies are under way to optimize the production of the glycol by the mutant.     

Enzyme immunoassay for the macrocyclic trichothecene roridin A: production, properties, and use of rabbit antibodies.

Antisera against roridin A were prepared by using a roridin A-hemisuccinate derivative coupled to human serum albumin as the immunogen. Antibodies could be detected in the sera of the immunized rabbits as early as 4 weeks after the initial exposure. After one booster injection at week 14, high antibody titers were measured over a period of 21 weeks. The specificity and sensitivity of the antibodies were tested by using roridin A-hemisuccinate coupled to horseradish peroxidase as an enzyme-linked toxin in a competitive assay with a double-antibody solid phase. The assay was most specific for the tested macrocyclic trichothecenes, and the relative cross-reactivities with roridin A, roridin J, verrucarin A, satratoxin H, and satratoxin G were 1, 0.41, 0.15, 0.15, and 0.07, respectively. When 16 nonmacrocyclic trichothecenes were tested, only diacetylverrucarol (0.0015) and verrucarol (0.0005) showed minor cross-reactivity. The sensitivity of the enzyme immunoassay for the detection of roridin A was in the range of 5 to 50 ng/ml (0.16 to 1.6 ng per assay).     

Conjugal transfer of group B streptococcal plasmids and comobilization of Escherichia coli-Streptococcus shuttle plasmids to Lactobacillus plantarum.

The antibiotic resistance group B streptococcal plasmids, pIP501 and pVA797, were conjugally transferred from Streptococcus faecalis to Lactobacillus plantarum. The Escherichia coli-Streptococcus shuttle plasmids, pVA838 and pSA3, were mobilized from S. sanguis to L. plantarum by pVA797 via cointegrate formation. pVA838 readily resolved from pVA797 and was present in L. plantarum as deletion derivatives. The pVA797::pSA3 cointegrate failed to resolve in L. plantarum.