Investigation of plasmid instability in amylase-producing B. subtilis using continuous culture.

Continuous culture was employed to study plasmid instability in an amylase-producing Bacillus subtilis 1A289 that was genetically manipulated. No true steady state could be obtained with 1A289(pEAA)-strain (plasmid)-due to its structural instability, which occurred both with glucose and Maltrin-100 as limiting carbon sources. The plasmid, pEAA (Cm(R), amy(+), i.e., chloramphenicol resistant, amylase positive) degenerated into a smaller plasmid, pEAA1 (CM(R), amy(-)) that was stable. There was a direct correlation between amylase-producing ability and this structural instability since f(amy) (fraction of cells with amylase-producing ability) reached zero at the same time that f (fraction of cells that are resistant to chloramphenicol) reached its maximum level. Since the deletion in pEAA was larger than the original amylase-gene insert, either all of part of the insert is absent from pEAA1. Though on discernible change in 1A289(pHV33), where pHV33 is the vector plasmid, was observed during continuous cultivation, its behavior was different from that of the stable 1A289(pEAA1).

Isolation and characterization of a polynucleotide phosphorylase from Bacillus amyloliquefaciens.

Bacillus amyloliquefaciens BaM-2 produces large amounts of extracellular enzymes, and the synthesis of these proteins appears to be dependent upon abnormal ribonucleic acid metabolism. A polynucleotide phosphorylase (nucleoside diphosphate:polynucleotide nucleotidyl transferase) was identified, purified, and characterized from this strain. The purification scheme involved cell disruption, phase partitioning, differential (NH4)2SO4 solubilities, agarose gel filtration, and diethylaminoethyl-Sephadex chromatography. The purified enzyme demonstrated the reactions characteristic of polynucleotide phosphorylase: polymerization, phosphorolysis, and inorganic phosphate exchange with the beta-phosphate of a nucleotide diphosphate. The enzyme was apparently primer independent and required a divalent cation. The reactions for the synthesis of the homopolyribonucleotides, (A)n and (G)n, were optimized with respect to pH and divalent cation concentration. The enzyme is sensitive to inhibition by phosphate ion and heparin and is partially inhibited by rifamycin SV and synthetic polynucleotides.

Inhibition of vesicular stomatitis virus replication by poly(2'-fluoro-2'-deoxyuridylic acid).

Poly(2'-fluoro-2'-deoxyuridylic acid) is known to be an effective inhibitor of the deoxyribonucleic acid polymerase found within the oncornaviruses. This synthetic polynucleotide was found to inhibit the replication of vesicular stomatitis virus in mouse L cells. The polymer was shown to be capable of inhibiting the viral ribonucleic acid (RNA)-dependent RNA polymerase, and it is proposed that this is the mechanism of antiviral activity. The following observations support this viewpoint: (i) the polymer is most active when added after virus adsorption; (ii) the antiviral activity is not species specific; and (iii) the polynucleotide is nontoxic to the host cell. Conventional methodologies designed to increase nucleic acid uptake by cultured cells do not show an increase in antiviral potency.