Adaptive responses of geobacter sulfurreducens to growth on formate during coculture and pure culture evolution

Submission note: Thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology to the College of Science, Health and Engineering, School of Life Sciences, La Trobe University, Victoria.

Geobacter sulfurreducens is an ‘electrogen’ of the order Deltaproteobacteria; meaning that it is both ‘electricigen’, donating electrons to insoluble electron acceptors extracellularly and ‘electrotroph’, receiving electrons from insoluble electron donors. This capacity for extracellular electron transfer (EET) is made possible through mechanisms utilizing c-type cytochromes and conductive pili. It resides in aquatic sediment environments and anaerobic wastewaters where it exists within microbial communities often containing other electrogens, acetogens and methanogens. Within these communities, a complex array of syntrophic and competitive interactions exists including the ability to respire through other microorganisms via direct interspecies electron transfer (DIET). Some of these microbial partners also interact indirectly through mediators such as formate and hydrogen through formate interspecies transfer (FIT) and hydrogen interspecies transfer (HIT). Furthermore, other IET interactions can occur via exogenous or endogenously produced redox mediators. The goal here was to characterize potential interactions between G. sulfurreducens and the microorganisms it is known to share environmental niches with. In particular its interaction with Pseudomonas aeruginosa, also an electrogen, was investigated as it differs in its EET mechanism by utilizing endogenously produced phenazine redox shuttles. Chapter 3 explores the syntrophic interaction discovered here between G. sulfurreducens and P. aeruginosa when grown in a formate and fumarate containing medium that induces their syntrophy. Providing formate as the sole electron donor, which is preferentially utilized by P. aeruginosa and fumarate as the sole electron acceptor and which can only be utilized by G. sulfurreducens, allowed for greatly enhanced growth of the cocultures compared to pure cultures of each in this medium. To understand whether. DIET was enabling this interaction, either via c-type cytochromes or phenazine redox shuttles, I also established cocultures with G. sulfurreducens ∆omcZ and ∆omcS c-type cytochrome mutants and P. aeruginosa ∆phz phenazine mutants. The outcome of these cocultures indicated that both OmcZ and OmcS cytochromes were necessary for the syntrophic interaction whereas phenazines were not. Due to the ca. 10 day long lag phase of the syntrophic cocultures, I progressed the cocultures through thirteen serial transfers (ca. 100 generations) to see whether the syntrophy could be enhanced further. This resulted in a shorter lag phase and decreased doubling time. SWATH-MS proteomics was performed on the cocultures to explore other potential mechanisms involved in the syntrophy. The results supported the initial involvement of cytochromes in the syntrophic interaction, however showed a progression toward diversification of IET mechanisms, toward HIT and formate utilization as the cocultures adapted. Therefore, a temporal distribution of DIET and HIT within the same coculture exists throughout evolution. In Chapter 4, I probe further into this intricate interaction through whole genome sequencing of both initial (subculture 0) and adapted (subculture 13) cocultures. Initial cocultures presented a small subpopulation of G. sulfurreducens containing a single nucleotide polymorphism (SNP) in the predicted tetR (GSU0951) gene. This mutation was strongly selected for as the cocultures adapted. TetR is a putative repressor of an operon containing CyaE and Resistance Nodulation and cell Division (RND) efflux pump proteins. SWATH-MS proteomics revealed that the expression of these operon encoded proteins mirrored the genetic frequency of the tetR SNP variant. This provides evidence for their predicted regulation within the TetR operon. Next, I devised and performed a spent medium testing protocol to determine if antibiotic production from P. aeruginosa may be causing the strong selection for increased expression of G. sulfurreducens RND multidrug efflux pumps. P. aeruginosa spent media had inhibitory action on Geobacter growth, supporting. its production of antibiotics. The evolved coculture spent medium however, did not inhibit Geobacter (previously unexposed to P. aeruginosa) growth indicating the presence of a stimulatory signal to encourage G. sulfurreducens proliferation in adapted cocultures. Inspection of the cocultures with fluorescence microscopy showed that G. sulfurreducens dominates both initial and adapted cocultures, and this dominance significantly increases with adaptive evolution. Hence, we see here that not only do the IET mechanisms evolve, but the dynamic from syntrophy to competition changes as well. Given the strong dominance of G. sulfurreducens in this formate and fumarate containing medium, it brings to question why it cannot grow well on its own. From the growth curves seen in Chapter 3, it is clear there is a low level of growth of wild-type DL1 strain of G. sulfurreducens in the medium. To test whether pili play a role in DIET, I had also established cocultures with P. aeruginosa and ∆pilA mutants of G. sulfurreducens. Surprisingly, not only did the ∆pilA mutants grow in coculture with P. aeruginosa, suggesting they are not necessary for DIET, but the ∆pilA mutant pure culture growth far surpassed that of DL-1 wild-type in this restrictive medium. In Chapter 5, I delve into exploring the reasons for why PilA-deficiency would lead to enhanced growth in these conditions. Formate is a C1 carbon compound and is deemed as a poor carbon source for G. sulfurreducens, which is not known to undergo the acetyl CoA pathway enabling C1 carbon assimilation. As with the coculture experiments, I attempted to coax this enhanced growth further via adaptive evolution through serial transfers. Both whole genome sequencing and SWATH-MS proteomics were performed on the initial and adapted ∆pilA pure cultures grown with formate and fumarate to explore the possible reasons for their unexpected growth. A strong increase in cytochromes was observed along with increased hydrogenase and ATP synthase protein abundance with adaptation of the cultures. Furthermore, overall biosynthetic pathways were also significantly increased. This, along with the low flux into the tricarboxylic acid (TCA) cycle suggest these cultures are boosting growth and biomass synthesis via proton motive force (PMF) driven energy production. The ∆pilA mutation is known to cause pleiotropic effects where cytochrome abundance is increased inside the cell but not in the extracellular matrix, since PilA is required for secretion of certain cytochromes outside the cell membrane. These cytochromes locked within the membrane confines may then act as additional electron carriers in the electron transport chain, enhancing the PMF generated and thus ATP produced.