Date of Award

2015

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Microbiology and Immunology

Abstract

Biofilms protect bacteria from environmental threats, including antibiotics; thus, biofilms formed during infections pose an increasing threat to human health. A natural model used to study biofilm formation in the context of a host is the symbiosis between Vibrio fischeri and its host, the squid Euprymna scolopes. Successful colonization depends on the formation of a biofilm and genes involved in making the polysaccharide matrix component, syp. In culture, biofilm phenotypes, including the formation of wrinkled colonies, similarly depend on syp. However, little is known about other factors that contribute to this phenotype. To expand the utility of currently available genetic tools, I developed a Tn5 transposon containing an outward facing lac promoter and a V. fischeri strain expressing lacI with which to control that promoter. To search for genes with previously uncharacterized roles in biofilm formation, I mutagenized the lacI-expressing biofilm-forming strain, and screened for mutants that failed to form wrinkled colonies. As expected, my screen for biofilm-defective smooth colonies yielded mutants of syp genes as well as other genes known to be required for biofilm formation. Several other mutants with disruptions in genes involved in central metabolism and electron transport we also isolated. Next, I shifted my attention to characterizing these mutant strains. Mutants lacking glnA, which encodes glutamine synthetase, exhibited a severe biofilm defect that could be rescued by the addition of glutamine, its product. A mutant defective for mdh (malate dehydrogenase) displayed an intermediate (diminished wrinkling) biofilm phenotype which could also be rescued by the addition of glutamine. Mutants lacking pck, which encodes PEP carboxykinase, also exhibited a severe biofilm defect, displaying not only a smooth colony phenotype but also adhering to the agar surface. I was unable to identify conditions that fully rescued the biofilm defect, but the addition of gluconeogenic carbon sources such as glucose abrogated the adherence phenotype. An sdhE mutant formed smooth colonies with small divots after prolonged growth, and an independently isolated sdhC mutant was similarly biofilm-defective; both mutants have defects in production of the succinate dehydrogenase complex. Of note was the phenotypes of three electron transport system mutants with insertions within two nqr (Na+-translocating NADH:ubiquinone oxidoreductase) genes and the ubiG (ubiquinone biosynthesis) gene: these mutants exhibited an extreme ability to strongly adhere attach to each other and/or the agar surface under biofilm-inducing conditions. The nqr phenotype required the production of Syp polysaccharide. Together, my work indicates a link between the metabolic state of the cell and biofilm formation.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

Download

Included in

Microbiology Commons

Share

COinS