Plasmid-mediated transformation in Bacillus megaterium.

A transformation system was developed for Bacillus megaterium by using antibiotic resistance plasmid deoxyribonucleic acid molecules derived from Staphylococcus aureus and Bacillus cereus. Lysozyme-generated protoplasts of B. megaterium allowed uptake of plasmid deoxyribonucleic acid in the presence of polyethylene glycol. Transformants expressed the antibiotic resistance determinants present on the plasmid deoxyribonucleic acid, and reisolated plasmid deoxyribonucleic acid yielded restriction endonuclease digestion patterns identical to those of the donor deoxyribonucleic acid.     

Carboxymethyl Cellulose Decomposition by Intestinal Bacteria of Cockroaches

The effect of freeze-drying on phenotypic reversion of amino acid auxotrophy to prototrophy was studied in Escherichia coli. In a radioresistant strain, E. coli H/r 30 (uvr+ exr+), which can repair the deoxyribonucleic acid damaged due to freeze-drying, an increased mutation frequency from auxotrophy to prototrophy was observed with increased time of freeze-drying of the cells. On the other hand, in a radiosensitive strain, E. coli NG 30 (recA), which cannot repair the damaged deoxyribonucleic acid due to a lack of repair enzyme system, no significant reversion occurred, although the survival rate was very low. The rate of phenotypic reversion dut to freeze-drying in both E. coli RIMD 0509109 (uvr+ exr+) and RIMD 0509115 (uvr exr+) was almost the same, indicating that the phenomenon is independent of the uvr character. From these results it is concluded that mutation was induced in E. coli cells during the rehydration when the damaged deoxyribonucleic acid was repaired by exr character of the cells. Thus, we propose that a serious consideration should be paid to the freeze-drying technique to preserve bacterial cells.     

Carboxymethyl cellulose decomposition by intestinal bacteria of cockroaches

The effect of freeze-drying on phenotypic reversion of amino acid auxotrophy to prototrophy was studied in Escherichia coli. In a radioresistant strain, E. coli H/r 30 (uvr+ exr+), which can repair the deoxyribonucleic acid damaged due to freeze-drying, an increased mutation frequency from auxotrophy to prototrophy was observed with increased time of freeze-drying of the cells. On the other hand, in a radiosensitive strain, E. coli NG 30 (recA), which cannot repair the damaged deoxyribonucleic acid due to a lack of repair enzyme system, no significant reversion occurred, although the survival rate was very low. The rate of phenotypic reversion dut to freeze-drying in both E. coli RIMD 0509109 (uvr+ exr+) and RIMD 0509115 (uvr exr+) was almost the same, indicating that the phenomenon is independent of the uvr character. From these results it is concluded that mutation was induced in E. coli cells during the rehydration when the damaged deoxyribonucleic acid was repaired by exr character of the cells. Thus, we propose that a serious consideration should be paid to the freeze-drying technique to preserve bacterial cells.     

Regulation of argA operon expression in Escherichia coli K-12: cell-free synthesis of beta-galactosidase under argA control.

Regulation of argA operon expression in Escherichia coli K-12 was studied in a cell-free, deoxyribonucleic acid-dependent, enzyme-synthesizing system. lambdaAZ-7 deoxyribonucleic acid, which carries a fusion of the lacZ structural gene to the argA operon so that beta-galactosidase synthesis is under argA regulation, was used as the template. To eliminate extraneous readthrough from lambda promoters, lambda repressor was introduced into the synthesis mixtures by preparing the S-30 component from a strain (514X5a-12-29) that carries a multicopy hybrid plasmid (pKB252) containing the lambdacI gene. Under these conditions beta-galactosidase synthesis was repressed 90% by the arginine repressor when a sufficient concentration of L-arginine was present. This repression could be overcome by escape synthesis when the lambdaAZ-7 deoxyribonucleic acid concentration in the synthesis mixtures was increased. Guanosine 3'-diphosphate-5'-diphosphate stimulated beta-galactosidase synthesis from this template.     

Growth inhibition of Escherichia coli by E colicin plasmids

Plasmids were isolated from E colicinogenic strains and transformed into prototrophic Escherichia coli K 12 strain DB364. Screening of E colicinogenic transformants for growth on defined medium revealed an apparent amino acid auxotrophy mediated by E4 and, to a lesser extent, E7 colicin plasmids. The auxotrophy was further investigated in E4 colicinogenic strains. From such auxotrophic transformants, denoted Pmi+ (plasmid-mediated inhibition of growth), Pmi- variants were obtained at a frequency of 3 X 10(-4) per bacterium. Plasmid loss was not detected among Pmi- clones. Isolation of E4 colicin plasmids from Pmi- clones and retransformation of strain DB364 with these plasmids showed that 40% of the plasmids were unable to inhibit growth of DB364 and were inferred to have alterations in an E4 colicin plasmid gene termed pmi. All such plasmids were indistinguishable from native E4 colicin plasmids, with respect to colicin immunity, colicin production and excretion, and sensitivity to lysis by mitomycin C. Experiments examining the nutritional basis of the plasmid-mediated auxotrophy indicated that at least seven amino acids, isoleucine, leucine, valine, arginine, methionine, serine and glycine, were involved in the auxotrophy. However, supplementation with only these seven amino acids did not completely restore growth. Assays of the activities of enzymes involved in amino acid biosynthesis in colicinogenic and non-colicinogenic strains under repressing and derepressing growth conditions suggested that E4 colicin plasmids did not repress synthesis of the implicated amino acids.     

Ti plasmid-controlled chromosome transfer in Agrobacterium tumefaciens.

In octopine-type A. tumefaciens R10, transfer of chromosomal arginine degradation genes (arc genes) was observed under conditions in which Ti plasmid transfer took place. However, transconjugants that had acquired the arc genes but not the Ti plasmid were recovered. During this process, several other chromosomal genes, such as genes encoding phage resistances or genes complementing a galactose utilization mutation or a glycine-serine auxotrophy, were transferred from strain R10 to the recipient.     

Methionineless Death in Escherichia coli

Methionine auxotrophs of strains derived from Escherichia coli 15 lose their colony-forming ability when deprived of this amino acid. Late addition of methionine to liquid cultures did not restore plating efficiency but permitted growth of surviving cells. This phenomenon, termed methionineless death (mld), was not observed with methionine auxotrophs of E. coli strains B, W, or K(12), nor was a similar amino acidless death observed with corresponding auxotrophs of E. coli 15 for arginine, tryptophan, proline, isoleucine, and leucine. Mld was not dependent upon the genetic site determining methionine auxotrophy, nor did it affect the decarboxylation of methionine or the stability of methionyl-transfer ribonucleic acid synthetase activity of starved cells. Death was not altered by the presence of spermine or spermidine but was abolished by the methionine analogue, alpha-methylmethionine. Simultaneous starvation of another amino acid in a multiple auxotroph also significantly reduced mld, suggesting a possible role of protein synthesis. The onset of mld is correlated with a lower net increase of deoxyribonucleic acid.     

Studies of colicin E1 plasmid functions by analysis of deletions and TnA insertions of the plasmid.

The further identification of regions of the colicin E1 plasmid that affect plasmid functions has been achieved by studying deletions and TnA insertions of the plasmid. Colicin production, colicin immunity, relaxation of plasmid deoxyribonucleic acid, and plasmid incompatibility functions have been examined. A strong correlation has been observed between the ability of colicin E1 plasmid deoxyribonucleic acid to be relaxed and the ability of that plasmid to be transferred by conjugation.     

Detection of plasmid deoxyribonucleic acid in an isolate of Lactobacillus acidophilus.

Eight strains of Lactobacillus acidophilus were examined for the presence of plasmid deoxyribonucleic acid, and one, a pig intestinal isolate, showed the presence of a 13.7- and a 6.3-megadalton plasmid. This is the first reported evidence for plasmid deoxyribonucleic acid in Lactobacillus acidophilus. The functions of these plasmids are presently unknown.     

Transformation of Salmonella typhimurium by Plasmid Deoxyribonucleic Acid

A modified transformation procedure that is effective for the introduction of plasmid deoxyribonucleic acid at high frequency into Salmonella typhimurium, as well as into Escherichia coli, is described. Transformed bacteria acquire a circular deoxyribonucleic acid species having the genetic and molecular characteristics of the transforming plasmid.     

Programming of poliovirus inhibition of deoxyribonucleic acid synthesis in HeLa cells

Ackermann, W. W. (The University of Michigan, Ann Arbor), and D. Wahl. Programming of poliovirus inhibition of deoxyribonucleic acid synthesis in HeLa cells. J. Bacteriol. 92:1051-1054. 1966.-Deletion of arginine from a culture medium reduced the rate of deoxyribonucleic acid (DNA) synthesis in uninfected HeLa cells. The normal rate was promptly restored by addition of arginine. Deletion of arginine also prevented poliovirus from inhibiting DNA synthesis in HeLa cells. However, the inhibitory potential of the infection and the capacity of the host cell for stimulation with regard to DNA synthesis were both retained in arginine-depleted cells which were infected. Upon addition of arginine late in the infection, DNA synthesis was first stimulated and then inhibited.     

Apparent Involvement of a Plasmid in Phaseotoxin Production by Pseudomonas phaseolicola

Three naturally occurring toxigenic strains (HB-36, G-50, and HB-33), one nontoxigenic strain (HB-20), and one ultraviolet light-induced toxinless mutant (G-50 Tox⁻) of Pseudomonas phaseolicola were examined by dye-buoyant density equilibrium centrifugation for the presence of plasmid deoxyribonucleic acid. All strains contained plasmid deoxyribonucleic acid. Comparison of the plasmid deoxyribonucleic acid of different strains by agarose gel electrophoresis showed that strain G-50 harbored three plasmids, whereas the rest of the strains contained two plasmids each. Irrespective of their toxigenicity, all strains shared the large-sized first plasmid band, but differed with respect to other plasmids. Restriction endonuclease analyses of the plasmids indicated that a 22.50-megadalton plasmid was common to two of the toxigenic strains (HB-36 and G-50). However, strain HB-33, which is also toxigenic, contained a much smaller plasmid (4.23 megadaltons). It is hypothesized that this small plasmid may have arisen by a recombination event from a larger plasmid.     

Plasmid deoxyribonucleic acid replication in Streptomyces griseus

A series of electron micrographs showing the presence of different molecular forms representing various replication stages of plasmid deoxyribonucleic acid from Streptomyces griseus was obtained. Based upon an analysis of these electron micrographs, a tentative model for plasmid deoxyribonucleic acid replication in S. griseus is proposed.     

Rp4 Promotion of Transfer of a Large Agrobacterium Plasmid Which Confers Virulence

Introduction of RP4 plasmid into Agrobacterium tumefaciens promotes the transfer on solid medium of large virulence-associated plasmids from virulent donor strains to a plasmidless avirulent recipient. Exconjugants were selected for the ability to utilize octopine or nopaline as the sole source of arginine, traits which are coded for by virulence-associated plasmids in the strains employed here. All exconjugants retained the arginine auxotrophy of the recipient strain, and were resistant to ampicillin and kanamycin, drugs to which RP4 confers resistance. Five exconjugant clones from one cross were shown by alkaline sucrose gradient analysis to contain both RP4 plasmid and the large virulence-associated plasmid of the donor strain. All five exconjugants exhibited virulence on carrot, sunflower and kalanchoe plants. These results indicate that virulence and the ability to degrade octopine are plasmid-borne traits in A. tumefaciens strains 15955 and A6, and extend the evidence that large plasmids in A. tumefaciens are vectors of virulence genes.     

The Rhizobium etli argC gene is essential for Arginine biosynthesis and nodulation of Phaseolus vulgaris

A Tn5-induced mutant strain (CTNUX5) of Rhizobium etli unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into an argC-homologous gene. Unlike its wild-type parent (strain CE3), the mutant strain CTNUX5 had an absolute dependency on arginine to grow. The argC gene was cloned from the wild-type strain CE3, and the resulting plasmid, pAR207, after transformation was shown to relieve the arginine auxotrophy of strain CTNUX5. Unlike strain CE3 or CTNUX5-pAR207, strain CTNUX5 showed undetectable levels of N-acetyl-gamma-glutamylphosphate reductase activity. Unless arginine was added to the growth medium, strain CTNUX5 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (nodulation factors) and to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris.     

Nature of transforming deoxyribonucleic acid in calcium-treated Escherichia coli.

A study of the reactivation of ultraviolet-irradiated plasmid and phage deoxyribonucleic acid molecules after transformation into Escherichia coli strains indicated that, when double-stranded deoxyribonucleic acid was used as the donor species, it was taken up without conversion to the single-standed form.     

Absence of Circular Plasmid Deoxyribonucleic Acid Attributable to a Genetic Determinant for Methicillin Resistance in Staphylococcus aureus

Plasmid deoxyribonucleic acid was not detected by centrifugal analysis of lysates of penicillinase-negative strains of Staphylococcus aureus harboring a determinant of methicillin resistance derived from strain Villaluz. When these strains contained a penicillinase plasmid, the plasmid deoxyribonucleic acid of methicillin-resistant and methicillin-susceptible strains was indistinguishable by the methods employed. The results indicate that the genetic determinant for methicillin resistance in the strains examined was not associated with a circular plasmid comparable to those that have been shown to determine resistance to benzylpenicillin, tetracycline, and chloramphenicol in S. aureus.     

Inadvertent gene silencing of argininosuccinate synthase (bcass1) in Botrytis cinerea by the pLOB1 vector system

For several years, researchers working on the plant pathogen Botrytis cinerea and a number of other related fungi have routinely used the pLOB1 vector system, based on hygromycin resistance, under the control of the Aspergillus nidulans oliC promoter and what was reported to be the β‐tubulin (tubA) terminator. Recently, it has been demonstrated that this vector contains a 446‐bp portion of the B. cinerea argininosuccinate synthase gene (bcass1) rather than the tubA terminator. As argininosuccinate synthase is essential for the production of l ‐arginine, inadvertent gene silencing of bcass1 may result in partial l ‐arginine auxotrophy and, indeed, may lead to altered phenotypes in planta. In this article, we report our findings relating to possible problems arising from this incorrect plasmid construction. As an absolute baseline, gene disruption of bcass1 was carried out and generated a strict auxotroph, unable to grow without exogenous arginine supplementation. The knockout displayed an alteration in host range in planta, showing a reduction in pathogenicity on strawberries, French bean leaves and tomatoes, but maintained wild‐type growth on grape, which is in accordance with the reported arginine availability in such tissues. Deliberate gene silencing of bcass1 mirrored these effects, with strongly silenced lines showing reduced virulence. The degree of silencing as seen by partial auxotrophy was correlated with an observed reduction in virulence. We also showed that inadvertent silencing of bcass1 is possible when using the pLOB1 vector or derivatives thereof. Partial arginine auxotrophy and concomitant reductions in virulence were triggered in approximately 6% of transformants obtained when expressing enhanced green fluorescent protein, luciferase, monomeric red fluorescent protein or β‐glucuronidase using the pLOB1‐based expression system, which inadvertently contains 446 bp of the bcass1 coding sequence. We recommend the testing of transformants obtained using this vector system for arginine auxotrophy in order to provide assurance that any observed effects on the development or virulence are a result of the desired genetic alteration rather than accidental bcass1 silencing.     

Partial characterization of nucleoids and nucleoid-plasmid complexes from Salmonella typhimurium.

Nucleoids from Salmonella typhimurium strain LT2 consist of supercoiled deoxyribonucleic acid structures that are ribonuclease labile sedimenting at 1,700S. More than 90% of the covalently closed circular deoxyribonucleic acid of a cryptic plasmid harbored by this strain cosediments with the host's 1,700S nucleoids.     

Mutants of the mini-F plasmid pML31 thermosensitive in replication.

Hydroxylamine mutagenesis was used for the induction of thermosensitive replication mutants of the mini-F plasmid pML31. Replication mutants were characterized by studying the segregation kinetics and the incorporation of [3H]-thymidine into plasmid deoxyribonucleic acid at the nonpermissive temperature. Based on these experiments two types of mutants could be distinguished. Mutants of type I are fast segregating with the kinetics expected if plasmid replication was blocked immediately. Double-label experiments showed a rapid shut-off of replication in these mutants at 42 degrees C. Mutants of type II segregate slower, showing only a partial inhibition of plasmid deoxyribonucleic acid synthesis at the nonpermissive temperature. The label incorporated at 42 degrees C was predominantly found in open circular plasmid molecules.