February 19, 2016

Identification, Induction, and Isolation of Bacteriophages Present in the Genomes of different Sinorhizobium Strains

Autumn Jensen '16

Nitrogen gas (N2) is the most abundant gas in the Earth’s atmosphere. Most living organisms areunable to utilize N2 and require ammonia for the biosynthesis of proteins and DNA. Bacteria of the genus Sinorhizobium convert N2 into ammonia through a process known as nitrogen fixation. These microbes live in symbiosis with important legume crops such as alfalfa, clover and peas. This symbiotic relationship is vital to agriculture because it provides a useful form of nitrogen for plants and reduces the use of chemical fertilizers. Bacteriophages are viruses that live in soils and infect nitrogen-fixing bacteria. Viral infection can reduce the numbers of bacteria available for nitrogen fixation and alter the symbiotic relationship with legumes. The objective of this research was to examine 51 previously sequenced Sinorhizobium strains and investigate the presence of dormant bacterial viruses (prophages) integrated into their genomes. We used a computational analysis system, PHAST, to search for likely prophages in Sinorhizobium DNA sequences. These analyzes showed that 13 out of 51 DNA sequences contained at least one intact prophage. The prophage-containing strains were subjected to environmental stressors such as UV light and mitomycin c to stimulate viral activation and excision from the host genome. These experiments lead to the isolation of active bacteriophages in 2 out of 13 strains, Sinorhizobium M102 and KH36b; no viral activation was observed in control experiments lacking the stressors. The M102 and KH36b viruses were purified and used to investigate their ability to infect 15 other Sinorhizobium strains. The data showed that the virus from strain M102 could only infect its original host, while the KH36b phage infected and effected growth of 6 of the strains tested. These results suggest that environmental stressors can activate dormant bacteriophages present in Sinorhizobium strains and potentially impact the populations of nitrogen-fixing bacteria in soils.

Detecting Antibiotic Resistant Soil Bacteria in Agricultural Areas

D.J. Cline '16

One of the major issues facing the world today is antibiotic resistant bacteria. As the use of antibiotics has become more widespread across the globe, so has the incidence of antibiotic resistance. The aim of this project was to determine if there is a difference in the percentage of tetracycline resistant bacteria in agricultural areas due to a potential use of tetracycline in feed for animals. To do this, we examined the amount of tetracycline resistant soil bacteria living in agricultural Minnesota compared to residential areas in Minnesota. We took soil samples from a number of Agricultural and residential sites and looked for known tetracycline resistance genes using PCR. We also grew cultures of soil bacteria on LB media and LB media with Tetracycline. We then calculated colony forming units per mL for each group to determine an approximate percentage of the tetracycline resistant bacteria living in the soil. The results seem to indicate that there are a higher percentage of tetracycline resistant bacteria in agricultural areas, however we believe that we will need to do further research with a larger sample size in order to find a statistical difference.