The spheroplast lysis assay for yeast in microtiter plate format.

A yeast lysis assay in the microtiter plate format improved precision and throughput and led to an improved algorithm for estimating lag time. The assay reproducibly revealed differences of 10% or greater in the maximal lysis rate and 50% or greater in the lag time. Clonal differences were determined to be the major source of variation. Microtiter-based assays should be useful for screening for drug susceptibility and for analyzing mutant phenotypes.     

Vegetative recombination of bacteriophage Mu-1 in Escherichia coli

Summary Twenty-two amber mutants of the thermoinducible mutator phage Mu-c4ts were isolated. These mutants fall into 11 complementation groups. The data obtained by crossing these amber mutants suggest that bacteriophage Mu-1 has a linear vegetative linkage map. In a recombination deficient host of the RecA type the recombination frequencies are extremely low, indicating that Mu-1, in contrast to many other E. coli phages, is dependent on the recombination system of its host. With as a helper phage, recombination between Mu phages in a RecA host is restored to about 1/3 of the frequency in a Rec⁺ host. Although Mu-1 is able to integrate efficiently into the chromosome of a RecA strain, it seems that its integration system does not contribute to vegetative recombination.The survival of UV-irradiated Mu-1 was measured on different radiation sensitive mutants of E. coli. The survival on a UvrB strain was very low as compared to the wild-type; the survival on a RecA strain was almost the same as on the wild-type.Research Fellow from the Laboratory of Genetics, State University, Leiden, The Netherlands.     

Thymineless bacteriophage induction in Staphylococcus aureus. I. High-frequency transduction with lysates containing a bacteriophage related to bacteriophage phi 11.

A thymine-requiring mutant of Staphylococcus aureus, strain 8325 (PI258)thy, undergoes prophage induction and lysis after thymine starvation. Four different phages were isolated from the lysate in low titers, among which was a phage designated phi 14, which differs from phage phi 11 in its immunity locus. The thymineless induced lysates of strain 8325(PI258)thy transduce the penicillinase plasmid at high frequency (10(-1), whereas transduction of chromosomal markers is inefficient. A plasmic-cured derivative of strain 8325(PI258)thy is also lysed by thymine starvation and be used for high-frequency transduction of other plasmids. Reconstitution of a strain of S. aureus that responds to thymine starvation was only partially successful, but this system can effectively be used to transduce plasmids or plasmid derivatives.     

Growth and survival of Azolla filiculoides in Britain I. Vegetative production

Azolla filiculoides Lam. causes serious weed problems in Britain, but its long-term survival might be limited by winter death. The aim of this study was to establish the low temperature responses and limitations of A. filiculoides sporophytes.
In the laboratory, normal vegetative growth was shown to continue at 5°C. Reddening of plants was a response to low temperature and high light conditions which could be prevented by shading. Adult plants died after short (18 h) exposure to −4°C but survived sub-zero temperatures >−4°C. Evidence was found of seasonal changes in chill tolerance, but not in freeze tolerance.
In outdoor culture, plants survived encasement in ice and air temperatures to −5°C. Additional evidence suggested that natural populations can readily survive air temperatures much lower than this. Microclimatic effects are likely to be responsible for this discrepancy between laboratory and outdoor culture results.
Three phenotyes were identified; survival, colonizing and mat forms.     

Study on the innocuity ofHirsutella thompsonii. I. Infectivity in mice and guinea pigs

The present work concerns the innocuity ofHirsutella thompsonii Fisher isolated in Cuba from a citrus rust mite,Phyllocoptruta oleivora Ashm. This phytopathogenic arachnide is an important pest of different citrus fruit plantations. The infectivity tests were performed in 220 animals (mice and guinea pigs) inoculated by intraperitoneal and subcutaneous injections. Animals showed macroscopic alterations such as nodules in the liver capsule, spleen, subcutaneous tissue; superficial fibrinous adhesions on these organs and hepatosplenomegaly. No fungi were recovered from organs at any time and only disintegrated hyphal elements were observed. The histopathological study disclosed a non-specific tissue reaction to a foreign body. The results obtained suggest the non-infectivity and innocuity ofHirsutella thompsonii Fisher in mice and guinea pigs.      

A mutation in Escherichia coli enhancing the UV-mutability of phage lambda but not of its infectious DNA in a spheroplast assay

Summary A mutant (called mul) has been isolated from E. coli AB1157 which increases up to 13 fold the rate of clear plaque mutations of extracellularly UV-irradiated phage . The mul-mutation does not affect the UV-mutability of T4rII mutants or various bacterial markers and therefore seems to act specifically on UV-irradiated phage . However, when spheroplasts prepared from mul cells were infected with either irradiated or unirradiated -DNA equal frequencies of clear plaques were produced. As there is indirect evidence (host cell reactivation) that the spheroplasts do not significantly exclude irradiated phage DNA it seems that the mul phenotype can be expressed only in complete cells.The mutation responsible for the mul phenotype has been mapped by conjugation and transduction to be located between the markers pyrE and ilv and probably marks a new gene. The mul mutation does not increase the UV-sensitivity of the cells nor does it affect their ability to perform host cell reactivation. The presence of a recA allel in a mul mutant abolishes the UV-mutability of phage .Part of this work is from the thesis of the first author in partial fullfilment of the requirements for the doctoral degree of the University of Bochum. Some of the results have already been presented at the 32. Kongreß für Hygiene und Mikrobiologie at Münster, 1969.     

Genetic recombination of bacteriophage T7 in vivo studied by use of a simple physical assay.

A new physical method was developed to assay genetic recombination of phage T7 in vivo. The assay utilized T7 mutants that carry unique restriction sites and was based on the detection of a new restriction fragment generated by recombination. Using this assay, we reexamined the genetic requirements for recombination of T7 DNA. Our results were in total agreement with previous findings in that recombination required the products of genes 3 (endonuclease), 4 (primase), 5 (DNA polymerase), and 6 (exonuclease). Recombination was found to be independent of DNA ligase and DNA packaging and maturation functions.     

Infectivity of different forms of lambda bacteriophage DNA in transfection of calcinated Escherichia coli]

Infectivity of linear lambdaDNA molecules is proved to be about a hundred times higher in calcinated E. coli K12 (lambai434) than in E. coli K12(lambda-): the levels of transfection were 1-3-10(7) and 1-2-10(5) infective centers per 1 mug DNA, respectively. In E. coli JC 5743 rec B21 defective for exonucleases I and V the level of transfection was 1-3-10(6). High infectivity of linear lambdaDNA in lysogenic cells cannot be explained by a helping effect of phage particles spontaneously liberated by these cells. It can be caused by recombinations of inserted lambdaDNA molecules with prophage or by the low activity of some nucleases in the lysogenic cells. Covalently closed and "Hershey" ring forms of lambdaDNA penetrate the calcinated cells as readily as linear molecules do but the infectivity of the former ones is proved to be very low.     

Helix hand fidelity in Bacillus subtilis macrofibers after spheroplast regeneration.

Left- and right-handed Bacillus subtilis macrofibers produced by strains FJ7 and C6D were converted to spheroplasts. Intact cells were regenerated and macrofibers were produced under conditions conducive for production of left- and right-handed structures. The resulting helix hand phenotypes always corresponded to those expected on the basis of the parental genotype.     

Enzyme activity and bacteriophage infection. I. Breakdown of adenosinetriphosphate

Experiments have been performed on the apyrase activity of E. coli, strain B. Although the dependence on pH and substrate is similar to that of rat tissue, the bacterial extracts are inhibited by Ca⁺⁺ and stimulated by Mg⁺⁺. In bacterial extracts the rate of phosphate release decreases in the course of the reaction, possibly owing to product inhibition. With multiple bacteriophage infection, the apyrase activity of the intact cells increased several fold, and the activity of extracts increased about 30 per cent. It is suggested that the changes could be attributed to an increase in the amount of enzyme although other alternatives cannot be precluded at present.