Part 1: Prophage induction has been demonstrated to be a sensitive indicator for a wide variety of toxic and mutagenic compounds and, as a consequence, has been utilized for biologically based carcinogen screenings. Fourteen marine bacterial isolates were screened for development into the Marine Prophage Induction Assay (MPIA), for marine samples. The selected isolate (P99-4S3) was identified by 16S rDNA sequencing as Pseudomonas aeruginosa. This isolate demonstrated a log-linear response to increasing dose of mutagens, and sensitivity to known environmental contaminants. Field-testing of the assay over two years demonstrated the MPIA would be a useful screening tool for environmental contamination. Part 2: The observed resistance of natural populations of Synechococcus to viral infection may be due to lysogeny with associated homoimmunity. A thirteen-month study of lysogeny in natural populations of Synechococcus demonstrated that lysogeny does occur and exhibits a seasonal pattern. Experiments were performed along a transect of the Mississippi River plume, which provided a variety of ambient nutrient regimes for comparison of lysogeny in Synechococcus. Nutrient amendments did not enable induction and often led to a decrease in viral production. Lysogeny in Synechococcus was primarily correlated with ambient host and cyanophage abundance. Cross-infectivity studies demonstrated cyanophage isolates possess variable virulence. The 35 isolates were examined by transmission electron microscopy (TEM), with 33 identified as myoviruses and two as podoviruses. This dominance of myovirus lytic cyanophage is consistent with prior observations. Twenty-five Synechococcus isolates were screened for prophage induction utilizing the inducing agent Mitomycin C. Eleven isolates demonstrated a statistically significant increase in virus-like particles (VLP’s) in treatment samples. No correlation was observed between their resistance to lytic viral infection and prophage induction. Isolate P99-14, with consistently high levels of prophage induction, was investigated further. In contrast to lytic cyanophage, the induced cyanophage is non-tailed. Differential staining and nuclease digestion experiments indicate that the induced particle contains single-stranded DNA. Environmental conditions potentially leading to prophage induction were investigated with Synechococcus cultures and natural populations. The isolate P99-14 demonstrated that high, continuous light caused prophage induction. Natural populations determined that shifts in salinity, temperature and phosphate are not triggers of prophage induction.

3-Methyl indole (3-MI) is a malodorant in food and animal waste and Clostridium scatologenes ATCC 25775 is the model organism for the study of 3-MI production. 3-MI is an anaerobic degradation product of L-tryptophan and can cause pulmonary disorders and death in cattle and goats. To elucidate the 3-MI biosynthesis pathway and the underlying genes, it is necessary to develop a system to allow genetic modification in Clostridium scatologenes ATCC 25775. Bacteriophages and transposons are useful tools to achieve this goal. Isolation of Clostridium scatologenes ATCC 25775 bacteriophage was attempted by prophage induction and enrichments using environmental sources. To induce prophages, cultures of Clostridium scatologenes ATCC 25775 were exposed to an effective concentration of mitomycin C at 2μg/ml and 5μg/ml. Induction with temperature was performed at 42ºC and 55ºC. Bacteriophage liberation, determined by a decrease in optical density was not observed in response to mitomycin C or by different growth temperatures. Nineteen environmental samples were tested for the presence of a bacteriophage that could infect Clostridium scatologenes ATCC 25775. The first cycle of enrichments suggested a decrease in cell density, consistent with the presence of a bacteriophage but this was not observed in further iterations. Plaque assays were performed to confirm the presence of phage, but no plaques were observed. Although, different experimental conditions were tested, a transducing bacteriophage capable of infecting Clostridium scatologenes ATCC 25775 was not isolated. Transposons have been successfully used to generate mutants in Clostridium difficle. Therefore, we attempted to introduce transposons Tn5 and Tn916 into Clostridium scatologenes ATCC 25775 using electroporation. Transposon mutagenesis using Tn916 did not yield antibiotic resistant colonies. In contrast, commercially available transposon Tn5 gave antibiotic resistant colonies. However, further screening of the colonies using transposon specific primers in PCR reactions, did not yield any PCR product. We were unsuccessful in developing a genetic modification system in Clostridium scatologenes ATCC 25775 using bacteriophage or transposons.

Viruses have an estimated global population size of 10 to the 31st, with a significant proportion found in the marine environment. Viral lysis of bacteria affects the flow of carbon in the marine microbial food web, but the effects of lysogeny on marine microbial ecology are largely unknown. In this thesis, factors that influence the control of lysogeny were studied in both the phiHSIC/Listonella pelagia phage-host system and in bacterioplankton populations in the Gulf of Mexico. Using macroarrays dotted with phiHSIC amplicons, viral gene expression over the course of a synchronous infection experiment was measured. Early, middle, late, and continually expressed genes were identified, and included open reading frames 45, 28, 18 and 17, respectively. Viral gene expression in cultures of the HSIC-1a pseudolysogen grown in low and normal salinity media was also analyzed. Overall, levels of viral gene expression were higher in the 39 ppt treatment as compared to the 11 ppt tre atment for most ORFs. In the 11 ppt treatment, free phage concentrations were one to two orders of magnitude lower than the 39 ppt treatment while intracellular phage concentrations were one-fold lower. Therefore, at low salinities, expression of phiHSIC genes is repressed resulting in a lysogenic-like state, while at 39 ppt, lytic interactions dominated. Few viral genes were highly expressed at low salinity, suggesting that repression of viral genes was controlled by host genes. Samples from the eutrophic Mississippi River Plume and the oligotrophic Gulf of Mexico were analyzed for lytic phage production and occurrence of lysogeny. Significant lytic viral production was only observed three stations, none of which were located within the MRP. This signifies that system productivity is not an accurate predictor of viral productivity. The lysogenic fraction was also inversely correlated to bacterial activity, which decreased with depth. These findings support the hypothesis that lysogeny is a survival mechanism for phages when host cell density is low or when conditions do not favor growth. A unifying theme from these experiments was that lytic processes dominated when bacterial growth conditions were optimal, while lysogeny was observed at unfavorable growth conditions or environmental stress (low salinity).