Genome based investigations towards understanding carbon source dependent bioelectrochemical activity of Pseudomonas aeruginosa strains

Berger, Carola; Agler-Rosenbaum, Miriam (Thesis advisor); Blank, Lars Mathias (Thesis advisor)

Aachen (2019)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2019

Abstract

The ubiquitously distributed Pseudomonas aeruginosa is able to generate electrical current in a bioelectrochemical system (BES). Here, the transmission of electrons from the cell to the anode is facilitated by self-produced redox active mediators, known as phenazines. These colored compounds are one of the hallmarks of P. aeruginosa and, if a sufficient concentration is reached, can be spotted by the naked eye on plates and in liquid cultures. During strain maintenance of the PA14 strain, individual colonies exhibiting strongly reduced levels of excreted phenazines were found. Depending on which carbon source was used, the isolate also showed a substantially altered time profile of, or overall decrease in phenazine production and current generation. Re-sequencing of the two colony forms verified a two base pair deletion in the LasR encoding gene, for the less phenazine producing variant. LasR is the master regulator of the complex quorum sensing (QS) system, regulating the expression of a myriad of different genes by direct and indirect means. Among these targets are the phenazine biosynthesis operons. This study proved that it is possible to correlate the carbon source dependent current generation of P. aeruginosa to the lasR gene and suggests a signaling link between 2,3-butanediol (2,3-BD) and the QS regulatory system in a lasR dependent manner. Besides the used carbon source, also the used strain of P. aeruginosa has proven to be of vast importance for its ability to facilitate current production in a BES. To help to understand what makes the natural BES isolate KRP1 a supreme candidate for further microbial fuel cell developments, its genome was de novo sequenced and analyzed in detail. Thereby it could be shown that KRP1 clearly is a P. aeruginosa variant, a relationship that was ambiguous before. Additionally, the overall genome organization of KRP1 was analyzed for the existence of genomic islands, hence islands of genes that are not shared by all members of the P. aeruginosa species. By the use of multiple software programs, 18 such regions could be determined, which sum up to about 10% of the whole genome. Many of the detected islands have been, in parts, reported in other P. aeruginosa strains. In this respect, KRP1 contains all genomic islands previously recognized in the highly virulent PSE9 variant and many of the islands responsible for increased virulence of the Liverpool Epidemic Strain (LES). This prompts the conclusion that the BES isolate KRP1 most likely is a highly virulent P. aeruginosa strain. The genome sequences of PA14 and KRP1, generated in this thesis, laid an ideal foundation to deeper investigate the regulatory networks causing the reported strain- and carbon source dependent current generation of P. aeruginosa in a BES. A whole-transcriptomic approach was chosen to generate a, as comprehensive as possible, connected picture of all intertwined pathways. To this end, individual RNA sequencing was performed for PA14 and KRP1 planktonic and biofilm cells, originating from a 2,3-BD- or glucose fed BES reactor. By generating individual libraries for each strain, subpopulation and carbon source, distinct differential gene expression analysis sets were possible in order to separate carbon source triggered alterations from changes caused by the different lifestyles. The transcriptional data of this study show that the biofilm subpopulation, formed at the medium-air interface on top of the reactor, is more active in phenazine production than the planktonic cells. Presumably, only a small fraction of the phenazine molecules diffuse into the media where they can actively be used for current generation. Indirect electron transfer with redox active phenazines requires multiple reduction and oxidation cycles of individual mediator molecules, but information about in vivo phenazine reduction in P. aeruginosa has been scarce. This study identified potential target enzymes that might facilitate unspecific phenazine reduction in vivo and explains why the carbon source 2,3-BD is reported to be utilized more rapidly than glucose, while at the same time experiencing greater catabolite repression than the hexose. Taken all results together, the individual investigations performed in the scope of this thesis help to understand the carbon source dependent bioelectrochemical activity of Pseudomonas aeruginosa strains.

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