Date of Award:

5-1-1981

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Biology

Department name when degree awarded

Life Sciences:Biology

Committee Chair(s)

Frederick J. Post

Committee

Frederick J. Post

Committee

Rex S. Spendlove

Committee

Larre Egbert

Abstract

Twelve strains of Halobacterium halobium from the national Research Council of Canada (NRC) and three extreme halophiles isolated from the Great Salt Lake were used as hosts in an attempt to obtain bacteriophages from the lake. We were successful with 7 NRC hosts and two of the lake hosts for a total of 8 (one was later lost) bacteriophages. All eight have proven to be DNA phages. As determined from melting point the GC ratios fell into three distinct groups: a low 48-51 group, an intermediate 54 group, a high 62-65 group, and a group of 3 phages with no melting point. This last group may be single stranded DNA although this needs further verification. Molecular weights are within the reported values for other bacteriophages. The latent period was somewhat variable ranging from 245 to 290 minutes with highly variable burst sizes ranging from 20 to 263. Exposure to the environmental stresses of heat, pH, ultraviolet light, and low salt resulted in no consistent groups of phages. Heat exposure indicated that all except one of the viruses were highly resistant to 70° C requiring in excess of 200 minutes to inactivate 109 or more particles. Extremes at 70° C were 4 minutes and more than 1000 minutes (estimated to be between 10,000 and 100,000 minutes). z values are within those reported for bacteria in general, 3.3° to 13.5° C except for L14 which behaved similarly to endospores with 22° C. Ultraviolet light exposure indicated two groups of phages based on relative D (decimal reduction) times. One group had low resistance, 0.11 to 0.22 hrs, while the other group was about 10 times more resistant. The more resistant group also showed a non-linear death curve. Generally a basic environment, up to pH 10.79, was less detrimental than an acid environment. There was also more variation between phages at higher pH's. Although all the halophages showed a 90% reduction time of about 0.17 hr (10 sec) at pH 0.87, several were relatively more resistant at pH 2.70. At least two groups were apparent when the viruses were removed to a salt water of low ionic strength. One group resisted 0.22% NaCl while the other group was readily inactivated. The host cells require 12% NaCl or more for growth. Below this level the cells lyse. While in this sense salt is required for virus infection of the host, the viruses may be more tolerant of low salt. Some may readily survive in seawater for example. The cross reactivity patterns of the halophages on the various hosts identified as H. halobium show some rather interesting patterns. Most of the halophiles in our collection have an optimum growth temperature of 40°-50° C and with some hosts the temperature of incubation of the host-virus system seems to be an important consideration. Phage 34004 would be monospecific except for this factor. Phages 34005 and 34007 exhibit the widest host spectrum infecting unclassified lake isolates as well as known H. halobium strains. The NRC cultures come from Africa, Israel, the Bahamas and Holland. In addition, HS15 is a strain of H. salinarium, HC23 is H. cutirubrum (NRC 34001), HT25 is H. trapanicum (NRC 34021), and HH24 is another H. halobium (NRC 34003). All of these react with one or more phages for H. halobium lending some support to the idea that there is only one species of Halobacterium. The lake strains of bacteria resemble H. halobiurn in some cases but not in others. L11 for example does not resemble H. halobium biochemically but does have other properties of that species. Since we only tried 12 separate strains as hosts, there are probably other viruses in the lake specific for those hosts which did not react. It will be interesting to see what these are like when isolated. In addition, the patterns of cross reaction suggest that these phages could be used in typing of the genus Halobacterium.

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