Thesis (M.S.)--University of Wisconsin--Madison, 1962. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Salvage of pyrimidine nucleosides and bases by S. griseus and the regulation of aspartate transcarbamoylase (ATCase) were studied. The velocity-substrate curve for S. griseus ATCase was hyperbolic for both aspartate and carbamoylphosphate. The enzyme activity was diminished in the presence of ATP, CTP, or UTP. The synthesis of ATCase was repressed in cells grown in the presence of exogenous uracil. The specific activity of cells grown with uracil was 43 percent of that for cells grown in minimal medium only. Maximal ATCase and dihydroorotase activities were found in the same column fraction after size-exclusion chromatography, suggesting that both activities could reside in the same polypeptide. The pyrimidine salvage enzymes cytosine deaminase and uridine phosphorylase were identified in S. griseus using HPLC reversed-phase chromatography.

The increasing emergence of antibiotic resistant strains of bacteria and fungi is driving the need to increase the production of current antibiotics and produce novel antimicrobial compounds. This study worked to increase the production of cycloheximide and streptomycin antibiotics by co-culturing Streptomyces griseus with other industrially important microbes. 1-3 industrial challenge microbes at a time were added to a culture of S. griseus and allowed to grow for one week in shake flask cultures before harvesting and quantifying antibiotic production. Fifteen different industrial challenge microbes placed in 35 different combinations were used in the study and 17 of these combinations were found to significantly increase antibiotic production after analysis with ANOVA. Antibiotic production was confirmed using bioautograms. Three of the successful different co-cultures were then subjected to a study to see when industrial challenge microbe addition was optimal. Results suggest that the optimal time to add the challenge microbes was 1-3 days following the original S. griseus inoculation. Dead challenge microbes were also added to a culture of S. griseus and it was found that these significantly increased cycloheximide as much as the live co-cultures did. / Department of Biology

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To further understand the ATCase/DHOase bifunctional complex formed in Streptomyces, the genes encoding these and other pyrimidine enzymes were identified and characterized. Polymerase chain reaction (PCR) was utilized in this effort. Primers were constructed by selecting conserved regions of pyrimidine genes from known gene and protein sequences of a wide variety of organisms. These sequences were then optimized to Streptomyces codon usage. PCR products were obtained from internal sites within pyrimidine genes and also from primer combinations of different genes. The size, orientation, and partial sequence of the resulting products shows that Streptomyces has a gene organization of pyrR followed by pyrB, pyrC, carA, carB, and pyrF in an operon similar to that found in other Gram-positive bacteria.

Bacteriophages, or simply "phages," are the most abundant biological entities on the planet and are thought to be the largest untapped reservoir of available genetic information. They are also important contributors to both soil health and nutrient recycling and have significantly influenced our current understanding of molecular biology. Bacteria in the genus Streptomyces are also known to be important contributors to soil health, as well as producing a number of useful antibiotics. The genetic diversity of large (> 30) groups of other actinobacteriophages, i.e. phages infecting a few close relatives of the Streptomycetes, has been explored, but this is the first formal effort for Streptomyces-infecting phages. Described here are a group of 45 phages, isolated from soil using a single Streptomycete host, Streptomyces griseus ATCC 10137. All 45 phages are tailed phages with double-stranded DNA. Siphoviruses predominate, six of the phages are podoviruses, and no myoviruses were observed. Notably present are seven phages with prolate icosahedral capsids. Genome lengths and genome termini vary considerably, and the distributions of each are in line with findings among other groups of studied actinobacteriophages. Interestingly, the average G+C among the 45 phages is around 11% lower than that of the isolation host, a larger disparity than reported for other groups of actinobacteriophages. Eighteen of the phages carry between 17 and 45 tRNAs and 12 of those carry a single tmRNA. Forty-three phages were grouped into seven clusters and two subclusters based on dot plot analysis, average nucleotide identities, and gene content similarities. Two phages were not clustered with other phages in this dataset. A total of 5250 predicted genes were sorted into 1300 gene "phamilies," with about 8% of the total phamilies having only a single member. Analysis of gene content among the 45 phages indicates first that most clusters presented here appear to be relatively isolated from one another, with phages in any one cluster generally sharing < 10% of their genes with phages in other clusters described here. Secondly, most of the phages here are more than twice as likely to share genes with phages isolated on bacteria outside of the genus Streptomyces than they are other phages isolated using a Streptomycete as host. These observations suggest that (1) the phage clusters here have a distinct extended host range, (2) those host ranges share overlap, and (3) Streptomyces griseus is likely not the preferred natural host for all phages described.

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Since phages use the host resources to replicate themselves after infection, the different sizes of the phage genome should influence the replication rate. We, therefore, hypothesized that the smaller genomes should burst the cell faster than the larger ones. As well, the shorter genomes would have greater burst sizes because they should replicate faster. Here, we obtained 16 phages of various genome length. All phages were isolated on Streptomyces griseus and available in our phage bank at the University of North Texas. We performed one-step growth studies for the 16 phages, as well as determined the host doubling time from its growth curve. The results show that S. griseus grown in nutrient broth has a doubling time of 5 hours and 22 minutes. This doubling time is used as a guideline for the phage growth studies. Because the filamentous nature of the host caused several difficulties during the experiment, we isolated single cells by sonication and centrifugation. After the cell number was determined by viable cell count, the cells were infected with each type of phage using a multiplicity of infection (MOI) of 0.5. The results show that phages' burst times range between 45 (±0, standard error) and 420 (±30) minutes and burst sizes from 12 (±0) to 1500 (±60) The statistical analyses show that there is no correlation between either genome size and burst time (R= -0.01800, P=0.97894) or genome size and burst size (R= -0.32678, P=0.21670). We further performed the comparative genomics studies to investigate whether the phages with similar burst times and burst sizes show similar genome structures. The studies show that Eddasa and Lorelei have similar burst times of 45 to 60 minutes and share 52 homologs. For burst size, only Tribute and Blueeyedbeauty that have similar burst sizes of 21-30, and they are genetically related because of the 48 shared homologs. Although this study did not find any correlation between genome size and burst time/burst size, it provides a foundation for further studies to determine what regulates these two traits.

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