Expression of a mineral phosphate solubilizing gene from Erwinia herbicola in two rhizobacterial strains.

A genetic construction was carried out using the broad host range vector pKT230 and plasmid pMCG898, which encodes the Erwinia herbicola pyrroloquinoline quinone (PQQ) synthase, a gene involved in mineral phosphate solubilization (mps). The final construction was transformed and expressed in Escherichia coli MC1061, and the recombinant plasmids were transferred to Burkholderia cepacia IS-16 and Pseudomonas sp. PSS recipient cells by conjugation. Clones containing recombinant plasmids produced higher clearing halos in plates with insoluble phosphate as the unique (P) source, in comparison with those of strains without plasmids, demonstrating the heterologous expression of the E. herbicola gene in the recipient strains. This genetic manipulation allowed the increase in mps ability of both strains, enhancing their potentialities as growth promoters of agricultural crops. These results represent the first report on the application of the recombinant DNA methodology for the obtaining of improved phosphate solubilizing ability from rhizobacterial strains for biofertilization purposes.

Isolation and enzymic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugar cane.

Acetobacter diazotrophicus, a nitrogen-fixing bacterium associated with sugar cane, secretes a levansucrase (sucrose-2,6-beta-D-fructan 6-beta-D-fructosyltransferase; EC 2.4.1.10). This enzyme is constitutively expressed and represents more than 70% of the total proteins secreted by strain SRT4. The purified protein consists of a single 58 kDa polypeptide with an isoelectric point of 5.5. Its activity is optimal at pH 5.0. It catalyses transfructosylation from sucrose to a variety of acceptors including water (sucrose hydrolysis), glucose (exchange reaction), fructan (polymerase reaction) and sucrose (oligofructoside synthesis). In vivo the polymerase activity leads to synthesis of a high-molecular-mass fructan of the levan type. A. diazotrophicus levansucrase catalyses transfructosylation via a Ping Pong mechanism involving the formation of a transient fructosyl-enzyme intermediate. The catalytic mechanism is very similar to that of Bacillus subtilis levansucrase. The kinetic parameters of the two enzymes are of the same order of magnitude. The main difference between the two enzyme specificities is the high yield of oligofructoside, particularly 1-kestotriose and kestotetraose, accumulated by A. diazotrophicus levansucrase during sucrose transformation. We discuss the hypothesis that these catalytic features may serve the different biological functions of each enzyme.

The 45-kb unit of major urinary protein gene organization is a gigantic imperfect palindrome.

The multigene family which codes for the mouse major urinary proteins consists of about 35 genes. Most of these are members of two distinct groups, group 1 and group 2. The group 1 and group 2 genes are organized in head-to-head pairs within 12 to 15 remarkably uniform chromosomal units or domains about 45 kilobase pairs (kb) in size. The 45-kb units are located on chromosome 4, and many of them are adjacent to each other. We propose that the 45-kb unit is a unit both of organization and of evolutionary change. In this study the homologies within the unit were observed by examining, in an electron microscope, heteroduplex and foldback structures made from cloned major urinary protein genes. These show that the 45-kb unit is a gigantic imperfect palindrome. Each arm of the palindrome contains two regions of inverted symmetry of 9.5 and 4.5 kb separated by a 3-kb nonsymmetrical region. We argue that the nonsymmetrical regions arose by a series of deletion events in the two arms of the palindrome. The center of the 45-kb unit is an 8-kb sequence without inverted symmetry flanked by the 9.5-kb regions, which contain the 4-kb genes and their immediate 5' and 3' flanking regions. The junction between adjacent 45-kb units is a 2- to 4-kb sequence without inverted symmetry flanked by the 4.5-kb regions. Some of the 45-kb units are arranged as direct tandem repeats. Others appear to be in inverted orientation with respect to a neighboring unit. Cloned major urinary protein genes show few incidences of the repetitive elements B1, B2, R, and MIF. Two elements, a B1 and an R, may be a constant feature of the 45-kb units. If so, in those cases in which the units are in tandem array, both of these elements will occur with a 45-kb periodicity. A comparison of corresponding parts of different 45-kb units shows that they differ because of a number of deletion or insertion events, particularly in the regions 3' to the genes.

Determination of the First Two Cleavage Furrows in Developing Eggs of Triturus Alpestris Compared with Other Forms: (cell cleavage=cytokinesis/furrow determination/Dettlaff units/newt eggs).

Eggs of Triturus alpestris were horizontally compressed at times before first and second cleavage in an experiment designed to measure the time at which the mitotic apparatus determines the direction of the subsequent cleavage furrow. The results showed that furrow determination was completed 0.46 Dettlaff units before the onset of furrowing. When a comparison was made of the times of furrow determination before cleavage in eggs of different animal groups using Dettlaff units, the results supported the idea that preparations for furrowing proceed differently in echinoderm eggs from eggs of sturgeon and amphibia.

The permeability to cytochalasin B of the new unpigmented surface in the first cleavage furrow of the newt's egg.

Two of 10 mug/ml cytochalasin B (CB) caused retraction of the first cleavage furrow in Triturus eggs, a spreading of the unpigmented surface from the furrow region and a flattening of the whole egg. CB appears to act against the contractility of the microfilamentous band at mid-cleavage so as to relax the furrow and also to weaken unpigmented surface to allow the egg to flatten. Uncleaved eggs and the initial formation of the cleavage groove were unaffected by CB. A fully-retracted first cleavage furrow reformed itself on transfer of the egg to normal medium but only at the time of second cleavage. Initiation of second cleavage depended upon there being sufficient of the original pigmented surface on the animal hemisphere. Tritium-labelled CB of high specific activty was prepared and used to study its ability to penetrate the surface of newt eggs during cleavage. Scintillation couting of whole eggs showed that CB was not taken into the newt egg until mid-cleavage (about 17 min after the double stripe stage) when new surface began to spread in the cleavage furrow. Fixation in glutaraldehyde and osmium tetroxide retained radioactivity in the egg, but more CB was retained after fixation in osmium tetroxide alone than after double fixation. Most of the retained radioactivity was in yolk platelets. Autoradiographs were prepared to sectioned eggs which had been fixed at late cleavage after [3H]CB had flattend the furrow. These showed that CB entered the egg through the unpigmented surface which formed in the furrow but it could not enter through the pigmented surface. The impermeability of the pigmented surface explains the observations that CB does not prevent initial furrowing at cleavage. Once inside the egg CB is transported slowly. CB penetrates to a limited extent beneath the pigmented surface from its border with the unpigmented surface in the first cleavage furrow and this seems insufficient in some circumstances to suppress the contractile phase of second cleavage.

Incorporation of tritiated cytochalasin B into ectodermal explants of the amphibian gastrula.

Ectodermal explants from gastrulae of Triturus alpestris were exposed to tritiated cytochalasin B (5 and 10 mug/ml) for 1/2-2 1/2 h. The flat explants failed to heal into compact spheres and the component cells gradually rounded up. Radioactivity in the fixed and sectioned material was analysed by light and electron microscopic autoradiography. Silver grains were found predominantly over the yolk platelets, but were also scattered over other cellular components. Areas containing pigment granules or the feltwork material, present in the apices of dissociating cells of the superficial layer, exhibited higher activity than areas containing mitochondria or cytoplasmic matrix. Lipid droplets and nuclei showed comparatively little activity. The results are discussed in relation to previous findings on the uptake of tritiated cytochalasin D by cell fractions.