Using Competition Assays to Assess Dormancy: Effects of Gene Knockouts on Growth in Micrococcus luteus

Dormancy is a survival growth state some bacteria enter under stressful environmental conditions, such as nutrient deprivation. Dormancy is characterized by a severe decrease in metabolic activity where bacteria become viable but not culturable (VBNC), which is measurable by plating and counting colony forming units (CFU). This decrease in metabolic activity also reduces the effectiveness of antibiotics as they are less able to enter cells. For this reason, understanding the mechanism of initiating, sustaining, and resuscitating bacterial dormancy is critical for improving treatment of bacterial pathogens utilizing dormancy to evade antibiotic treatment. Competition experiments can measure the relative fitness of bacterial populations by co culturing the two (or more) populations and allowing them to compete for the same resources. My goal was to use a simple competition assay to measure the relative fitness of wild-type Micrococcus luteus to gene knockout strains of genes suspected in the mechanism of dormancy, such as the uspA616 gene. First I demonstrated that the pigment synthesis gene, crtE, in M. luteus is a neutral site in both nutrient rich and nutrient poor media. Knockout of the crtE gene ([delta] crtE::kan) produced white bacterial colonies, as opposed to yellow colonies in wild-type M. luteus. The [delta] crtE::kan bacterial strain was found to have similar fitness to wild-type and was therefore used as an easily identifiable wild-type substitute in all other competition experiments. With the white [delta] crtE::kan M. luteus strain I then show that the UspA616 gene knockout ([delta] uspA616::kan) strain has similar fitness to wild-type M. luteus in nutrient rich media, but is significantly less fit in nutrient poor media.

Dormancy is a survival growth state some bacteria enter under stressful environmental conditions, such as nutrient deprivation. Dormancy is characterized by a severe decrease in metabolic activity where bacteria become viable but not culturable (VBNC), which is measurable by plating and counting colony forming units (CFU). This decrease in metabolic activity also reduces the effectiveness of antibiotics as they are less able to enter cells. For this reason, understanding the mechanism of initiating, sustaining, and resuscitating bacterial dormancy is critical for improving treatment of bacterial pathogens utilizing dormancy to evade antibiotic treatment. Competition experiments can measure the relative fitness of bacterial populations by co culturing the two (or more) populations and allowing them to compete for the same resources. My goal was to use a simple competition assay to measure the relative fitness of wild-type Micrococcus luteus to gene knockout strains of genes suspected in the mechanism of dormancy, such as the uspA616 gene. First I demonstrated that the pigment synthesis gene, crtE, in M. luteus is a neutral site in both nutrient rich and nutrient poor media. Knockout of the crtE gene ([delta] crtE