Beyond Plastic Degradation: Microbial Sensing and Response of Vibrio sp. to PET Breakdown Products within the Plastisphere

Abstract

Plastic production and the resulting waste significantly threaten the environment and numerous ecosystems. Due to increasing production rates, global plastic pollution is also rising rapidly. Plastic particles accumulate in a wide range of habitats, and nowadays are found all around the globe. These particles are rapidly colonized by diverse microorganisms. While microbial colonization offers potential advantages, such as contributing to plastic degradation, it also presents risks, particularly due to the accumulation of potentially pathogenic bacteria on plastic surfaces. Another major consequence of microbial colonization is biofouling, where bacteria adhere to plastic materials, used in various industries, leading to surface damage and reduced material durability. This thesis encompasses three chapters. In chapter one, this study gives for the first time insights into molecular keys, involved in Vibrio gazogenes polyethylene terephthalate (PET) colonization and subsequent polymer degradation, using deep transcriptomics, advanced imaging analyses and analytic technologies. The first part of the study focuses on the analysis and transcriptional profiling of the PET6 gene expression. PET6 is a recently discovered PETase in the marine bacterium Vibrio gazogenes DSM 21264. Within 24 hours of incubation, degradation products such as bis(2-hydroxyethyl) terephthalate (BHET), mono(2-hydroxyethyl) terephthalate (MHET) and terephthalic acid (TPA) were detected, when DSM 21264 was incubated with PET foil or powder. For a deep understanding of bacterial response in the presence of PET and its degradation products BHET and TPA, focusing on the transcription of the pet6 gene, multiple RNAseq approaches were conducted. Further, a number of natural substrates like chitin and cellulose were used, to study the response of DSM 21264 and its gene expression profile. Interestingly, the incubation of DSM 21264 in biofilm condition on PET vs PE surface or planktonic samples with PET powder did not increase the transcription of the pet6 gene. Highest transcription, however, was detected in planktonic samples, supplemented with BHET. Notably, the intermediate BHET significantly influenced bacterial gene expression on a global level by disrupting key signaling systems like quorum sensing (QS), c-di-GMP and CRP-cAMP signaling pathway. Using these second messenger bacteria adapt to their environment, regulating numerous metabolic pathways. This resulted in failure to form biofilms, altered colony morphology, and inhibited the biosynthesis of the red pigment prodigiosin. These findings imply that microbial and enzyme driven plastic degradation and the generated metabolites, thereby, may not only serve as carbon sources but also as potential signaling molecules. Furthermore, with this work I provided evidence that UlaG, which has been described as a highly promiscuous esterase, involved in ascorbate metabolism under anaerobic conditions, plays a significant role in BHET degradation. The transcription was 7.9-fold upregulated in the presence of BHET, and biochemical tests using recombinant UlaG verified that the Vibrio gazogenes UlaG is involved in BHET metabolism. Further work will have to evaluate its role in the plastisphere. In the second chapter, the colonization behavior of the Gram-negative bacterium Kaistella jeonii, native host of PET 30, was observed on PET surface, using Laser scanning microscopy (LSM). Kaistella jeonii is a member of the phylum Bacteroidetes and wide spread in nature. It plays an important role in the plastisphere. Thereby, my imaging analyses contributed to the characterization of the first PET-degrading enzymes from the phylum Bacteroidetes. In chapter three, Vibrio campbelli, a pathogenic member of the plastisphere, was analyzed on treated and untreated polyethylene (PE) foils with the aim to prevent biofouling. A novel surface treatment with poly-N-oxides grafted onto PE surface revealed significantly less bacterial colonization. Altogether, this study investigated for the first the influence of PET degradation products on the microbial community of the plastisphere, and offers solutions towards biodegradation and biofouling.