Biosynthesis of edeine: II. Localization of edeine synthetase within Bacillus brevis Vm4.

Edeine-synthesizing polyenzymes, associated with a complex of sytoplasmic membrane and DNA, were obtained from gently lysed cells of Bacillus brevis Vm4. The polyenzymes-membrane-DNA complex, isolated from dells intensively synthesizing edeines (18--20 h culture) contained edeine B. Edeine B was found to be bound covalently t o the edeine synthetase. The amount of edeine bound to polyenzymes was 0.1--0.3 mumol/mg protein, depending on the age of cells. Detachment of deeine synthetase with a covalently bound edeine B from the membrane-DNA complex was accomplished by a treatment with (NH4)2-SO4 at 45--55% saturation or by DEAE-cellulose column fractionation. In contrast to other components of the complex, the edeine-polyenzymes fragment was not adsorbed to the DEAE-cellulose. Sephadex G-200 column chromatography separated the edeine-polyenzymes complex into 3 fractions. Edeine-polyenzymes complex, obtained from lysozyme-Brij-58-DNAase treated cells, contained edeine B bound to two protein fractions of mol. wt 210 000 and 160 000. Edeine-polyenzymes complex detached from the complex with the membrane and DNA contained edeine B, bound only to protein fraction of mol. wt 210 000. Edeine A was not found in the edeine-polyenzymes complex. No accumulation of free antibiotics within 16--22 h old cells of B. brevis Vm4 was detected. The edeine-polyenzymes complex associated with the DNA-membrane complex has shown no antimicrobial activity. By treating of above with alkali, edeine B of specific activity: 80 units/mjmol was released. The complex of DNA-membrane associated with edeine-polyenzymes complex was able to synthesize DNA, under the conditions described for synthesis, directed by a DNA-membrane complex. Edeine when associated with this complex did not effect the DNA-synthesizing activity.     

Inactive form of edeine in the edeine-producing Bacillus brevis Vm 4 cells.

1. Exogenous edeine inhibits the synthesis of DNA and protein, but not that of RNA, in extracts of edeine-producing Bacillus brevis Vm 4 cells. This is analogous to the effect of edeine on extracts obtained from edeine-sensitive cells. 2. Producer cells, in contrast to sensitive ones, are not permeable to exogenous edeine. DNA synthesis in producer cells rendered permeable by toluene treatment becomes sensitive to edeine. 3. No free edeine could be detected in post-log producer cells during maximal synthesis of edeine. Nascent edeine exists in the cell in a biologically inactive form, bound to a fast-sedimenting fraction. Edeine B, identical to the antibiotic present in the medium, is released from this fraction by mild treatment with alkali.     

Inactive form of edeine in the edeine-producing Bacillus brevis Vm 4 cells

• 1.1. Exogenous edeine inhibits the synthesis of DNA and protein, but not that of RNA, in extracts of edeine-producing Bacillus brevis Vm 4 cells. This is analogous to the effect of edeine on extracts obtained from edeine-sensitive cells.
• 2.2. Producer cells, in contrast to sensitive ones, are not permeable to exogenous edeine. DNA synthesis in producer cells rendered permeable by toluene treatment becomes sensitive to edeine.
• 3.3. No free edeine could be detected in post-log producer cells during maximal synthesis of edeine. Nascent edeine exists in the cell in a biologically inactive form, bound to a fast-sedimenting fraction. Edeine B, identical to the antibiotic present in the medium, is released from this fraction by mild treatment with alkali.

     

Modification of glutamine synthetase in Bacillus brevis AG 4

The various forms of glutamine synthetase obtained from Bacillus brevis have been found to be antigenically identical. Alkaline phosphatase treatment of the fast moving form (GS4) reduced the electrophoretic mobility of the enzyme. Radiolabelling and autoradiographic studies have also indicated that 32P-incorporation is high in the form depicting high Rm value. Thus, it appears that these forms arise due to covalent modification of the enzyme involving a phosphate group.     

Multiple forms of glutamine synthetase in Bacillus brevis AG 4

Glutamine synthetase in Bacillus brevis AG 4, a Gram-positive spore forming bacteria, has been found to exist in multiple molecular forms. It was purified to electrophoretic homogeneity by single-step Blue Sepharose affinity chromatography. The native enzyme has a molecular weight of 600,000 with subunits of 50,000. The enzyme samples purified from different stages of growth differed in Mg2+ sensitivity and other kinetic properties. Four different enzyme samples selected on the basis of Mg2+ sensitivity showed distinct mobilities at pH 6.3 on PAGE using discontinuous buffer system. A correlation amongst Mg2+ sensitivity, electrophoretic mobility, and kinetic properties was highly suggestive of multiple forms of glutamine synthetase in Bacillus brevis arising due to modification.     

Oxygen-dependent inactivation of gramicidin S synthetase in Bacillus brevis.

Incorporation of L-[14C]ornithine into gramicidin S by crude, unfractionated lysozyme extracts of Bacillus brevis ATCC 9999 was shown to represent the activity of the gramicidin synthetase complex. Frozen-thawed cells were the source of active extracts, but when cells were shaken in air at 37 degrees C, they rapidly lost activity in a first-order reaction with a half-life of 13 min. Protease inhibitors and inhibitors of energy metabolism had no effect on the inactivation process in frozen-thawed cells. Stabilization was achieved when the cells were shaken in nitrogen or helium instead of air. The addition of dithiothreitol produced a moderate degree of stabilization. The L-ornithine- and D-phenylalanine-activating activities of the gramicidin S synthetase complex were also lost during aeration of the cells. Crude cell-free extracts also lost activity when they were shaken in oxygen, but, in this case, inactivation was slower (half-life of 80 min). Nitrogen also stabilized these cell-free extracts.     

Detection of two forms of glutamine synthetase in Bacillus brevis (A. G. 4)

A laboratory isolate of $\text{Bacillus}\text{brevis}$ could grow and sporulate on an amino acid, viz., alanine or glutamate or aspartate as single source of carbon and nitrogen. It failed to sporulate if the amino acid was replaced by the corresponding keto acid and ammonium sulphate in the medium, although, normal growth was observed. One of the key enzymes in nitrogen assimilation, the glutamine synthetase, has been purified by DE-52 and affinity column chromatography from both alanine and pyruvate grown cells. The kinetic and other properties of both of these enzymes were studied. The enzyme isolated from alanine grown cells differed significantly from that isolated from pyruvate grown cells (viz.,pH optima, response to Mg⁺⁺ and other effectors). A possible role of glutamine synthetase in the initiation of bacterial sporulation is discussed.     

Biosynthesis of isotopically labeled gramicidins and tyrocidins by Bacillus brevis

The three-dimensional structure of bilayer-associated gramicidin A is available from a structural data base. This and related peptides are, therefore, ideal model compounds to use during the implementation and development of new NMR techniques for the structural investigations of membrane proteins. As these methods rely on the isotopic labelling of single, selected or all sites, we have, investigated and optimised biochemical protocols using different strains of the Gram-positive bacterium Bacillus brevis. With newly developed schemes for isotopic labelling large amounts of gramicidin and tyrocidin enriched with stable isotopes such as ¹⁵N or ¹⁵N/¹³C have been obtained at low cost. A variety of analytical and spectroscopic techniques, including HPLC, mass spectrometry and NMR spectroscopy are used to characterise the resulting products.     

Two enzymically active forms of glycyl-tRNA synthetase from Bacillus brevis. Purification and properties.

Using sucrose density centrifugation and gel filtration of a 105000 X g supernatant of Bacillus brevis two enzymic activities of glycyl-tRNA synthetase were separated. Enzyme catalyzing the aminoacylation of tRNA (E1) elutes in a high-molecular-weight region. Enzyme active in glycylhydroxamate formation (E2) elutes from a Sephadex gel column and sediments in sucrose density gradient in a region of relatively low molecular weight. The presence of two enzymic activities does not depend on the method of cell disruption; their proportion does not change when protease inhibitor (diisopropylphosphorofluoridate) is added to the extraction buffer. Both E1 and E2 were purified to a nearly homogeneous state. Sedimentation coefficients (sw,20) were found to be 8.6 S and 3.6 S and molecular weights 226000 and 66000 for E1 and E2, respectively. During storage, E1 dissociates into two components, one of which has electrophoretic mobility identical to E2. The molecular weight of the other component is about 1600000. Electrophoresis of E1 in the presence of sodium dodecylsulfate reveals two bands corresponding to molecular weights of 81000 and 30000. Under these conditions, E2 dissociates into a polypeptide with a molecular weight of 30000. Valine was found to be the N-terminal amino acid for E2 and both valine and glutamic acid were N-terminal amino acids for E1. It is concluded that E1 is a tetrameric protein consisting of two large and two small subunits (alpha2beta2). E2 is a component of E1 with a structural formula alpha2.     

Purification and properties of guanosine 5', 3'-polyphosphate synthetase from Bacillus brevis.

A ribosome-independent guanosine 5',3'-polyphosphate synthetase has been highly purified from Bacillus brevis (ATCC 8185). The enzyme has a molecular weight of 55,000, as measured by sucrose density gradient centrifugation. Like the ribosome-connected stringent factor of Escherichia coli, it catalyzes the synthesis of the guanosine 5', 3'-polyphosphates by a pyrophosphoryl transfer mechanism from adenosine triphosphate (ATP) to guanosine di- or triphosphates (GDP, GTP). It has an apparent Km of 0.14 mM for GDP and 0.77 mM for GTP, and is specific for the guanosine ribonucleotides as pyrophosphoryl acceptors. Several ATP analogues were tested for their ability to donate the pyrophosphoryl group. Mg2+ was required as a counter ion for the nucleotide substrate; however, an excess of Mg2+ was inhibitory. The property of the B. brevis enzyme is compared with the ribosome-linked enzyme of E. coli and an extracellular enzyme excreted by several types of Streptomyces reported upon recently.