Bacteria, while traditionally studied in liquid suspensions, are often found in nature as biofilms, aggregates of cells enclosed in self-produced matrices. Cells in biofilms have a fitness advantage over those that are free-living, as the biofilm lifestyle is correlated with increased resistance to various assaults, including antimicrobials, UV exposure, and dehydration. These biofilm-associated characteristics have important clinical implications, as biofilm-based bacterial infections are a major cause of morbidity in immunocompromised individuals. With this increased resiliency, however, comes a major challenge that arises during biofilm growth: the formation of resource gradients. My thesis work focused on one particular gradient, that of oxygen, which is established in biofilms formed by Pseudomonas aeruginosa. This bacterium has multiple mechanisms for coping with limited access to oxygen, including a highly-branched respiratory system for optimal oxygen scavenging and production and utilization of redox-active molecules called phenazines. The purpose of this thesis has been to investigate the different strategies used by P. aeruginosa to deal with the oxygen limitation precipitated by the biofilm lifestyle.
In Chapter 1, I will provide the necessary background for understanding the principles of redox balancing, metabolism, respiration, biofilm physiology, and phenazine utilization in P. aeruginosa. The work described in Chapter 2 provides evidence for the formation of a novel terminal oxidase complex that plays a biofilm-specific role in P. aeruginosa growth. The results in this chapter also suggest that specific terminal oxidase complexes differ in the timing of their contributions to biofilm growth and implicate the novel complex in mediating reduction of phenazines in biofilms.
Chapter 3 expands upon the principle of metabolic versatility exemplified by the results discussed in Chapter 2. The research presented in this chapter looks at how varying the source of electrons that feed into the respiratory chain influences downstream electron transfer steps, including terminal oxidase activities and phenazine production and utilization. The data presented in Chapters 2 and 3 add to the growing body of evidence that bacterial growth in liquid culture is distinct from that in biofilms and underscores the need for more biofilm-based research that can inform treatment strategies for P. aeruginosa infections.
The results described in Chapter 4 take an even broader look at the strategies used by P. aeruginosa to sustain efficient metabolism under conditions of potential stress. An important node of central metabolism is pyruvate, which can be transformed in a number of ways. In this chapter, I will consider two pathways of pyruvate metabolism: fermentation to lactate and carboxylation to oxaloacetate. I will present data indicating that a previously-uncharacterized lactate dehydrogenase contributes to P. aeruginosa growth under specific growth conditions and that pyruvate carboxylation contributes to optimal progress through central metabolic pathways. I will also describe experiments that characterize the contributions of another carboxylase, previously thought to function as the pyruvate carboxylase, to P. aeruginosa’s ability to grow on selected nutrient sources. Finally, I will discuss how redox state informs biofilm formation in a phylogenetically distinct bacterium, Bacillus subtilis, highlighting the universality of redox reactions in driving metabolic processes.
In sum, the research presented in this thesis broadens our understanding of the immense respiratory and metabolic flexibility of P. aeruginosa and serves as an important reminder of the discrete factors that govern liquid culture and biofilm growth.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/d8-77tr-1g05 |
| Date | January 2018 |
| Creators | Jo, Jeanyoung |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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