Graduate Thesis Or Dissertation
 

Genetic homology and exchange in lactic acid streptococci

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  • The dairy industry relies primarily on consistent acid production by the lactic streptococci for the manufacture of certain cheeses and fermented dairy products. Variation in these cultures due to genetic exchange has not been thoroughly investigated. This study was undertaken to determine if genetic homology exists within the lactic group, and if genetic exchange by transformation could be demonstrated. Thermal denaturation curves of deoxyribonucleic acid isolated from several strains of Streptococcus lactis, Streptococcus cremoris and Streptococcus diacetilactis showed average mean Tm values of 84.1°C, 84.4°C and 83.7°C, respectively. These values corresponded to guanine plus cytosine percentages of 36.1, 36.8 and 35.1 for the three species. Tm values determined, for comparitive purposes, for species of streptococci belonging to the pyogenic, viridans and enterococcus groups revealed values comparable to those found for the lactic streptococci. The values ranged from 84.0°C for Streptococcus agalactiae to 86.2°C for Streptococcus salivarios, but were clustered around 84.0°C. On the assumption that the molecular distribution of bases within the DNA might be gaussian, normal probability graph paper was used to determine the Tm value, which was the 50 percent point. Comparison of this method with the usual graphical method of determining the Tm value showed that nearly identical results could be obtained. Results from the probability graph paper indicated that the distribution curves derived from thermal denaturation data for some samples were skewed to the left at the lower temperatures of melting. It was found that the skewed portion of the distribution curve could be resolved into two normal distribution curves that overlapped one another; the skewed portion formed a smaller distribution at the lower temperatures. Cesium chloride density gradient factionation of DNA from strains showing skewed distribution curves indicated that a fraction could be separated from the main portion of the DNA. The smaller portion corresponded to a fraction of lower density, the presence of which had been predicted from the distribution curves. A transformation system for S. diacetilactis 18-16 was established, using streptomycin resistance as the marker. The frequency of transformation was low, but the time of appearance of transformants occurred consistently within three to five hours of incubation in the competency medium. At 21°C, the time of appearance of transformants was delayed for five hours; the number of transformants then rose slowly until the experiment was terminated. At 30°C the number reached a maximum at the five-hour interval, and then declined to the end of the experiment. The frequency of transformation was increased by concentrating the cells. Filtration of the samples before exposure to the donor DNA proved to be a more efficient method of cell concentration than centrifugation. The time of maximum expression of the incorporated streptomycin- resistance marker was found to be eight hours at 30°C. The transfer of mannitol utilization by transformation to a mannitol mutant of S. diacetilactis 18-16 was determined qualitatively, and the time of appearance of transformants corresponded to that found for the streptomycin-resistance marker. Minimum methylene blue adsorption by competent cultures occurred at four hours of incubation. This interval corresponded to the midpoint of the range of time at which transformants were detected in transformation experiments.
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file details KnittelMartin1965.pdf 2017-08-04 Pubblico Scaricare