Antimicrobial potential of Geobacillus sp. ZGt-1 isolated from Zara hot spring in Jordan : Piecing the puzzle of the antagonistic activity of a novel bacterial strain

University dissertation from Biotechnology, Lund University

Abstract: The problem of antibiotic resistance is rising continuously, creating a warning signal that calls for finding alternatives. Not only that, but there is also a rising demand for natural food preservatives. The pharmaceutical and food industry sectors are working on fulfilling these needs. This thesis is exploring possible solutions to these issues. The research studies presented here introduce the potential of thermophilic bacteria isolated from hot springs as a source for antimicrobial agents that could be applied in the pharmaceutical and food industries. The focus is on the bacterial strain Geobacillus sp. ZGt-1 that was isolated from Zara hot spring in Jordan. Experimental work and in silico analyses of the genome sequence of this strain revealed its antimicrobial potential. This strain grows at 60 °C and antagonizes the growth of the food spoiling thermophilic bacterium Geobacillus stearothermophilus. It also antagonizes the growth of the mesophilic bacteria Bacillus subtilis and the pathogenic Salmonella Typhimurium, both grown at 37 °C. The thesis presents antimicrobial peptide and protein candidates of the strain ZGt-1. These candidates include a list of secreted proteins within the range of 10–30 kDa that are thermostable and SDS-resistant. They also include a putative novel lanthipeptide, which we identified as Z-geobacillin that is smaller than 3.5 kDa. The candidates also include toxins belonging to various families of type II toxin-antitoxin system, within the range of 3–17 kDa.The protein candidates were produced at 60 °C by immobilizing the cells of ZGt-1 in agar beads that were cultivated in sequential batches to solve the issue of producing the proteins in liquid. The proteins were then purified and identified using a combination of proteomic and bioinformatic tools.The Z-geobacillin represents the first lanthipeptide identified in a hot spring-inhabiting bacterium and is expected to be more stable than nisin. In addition to Z-geobacillin, seven putative novel class-I lanthipeptides were predicted to be produced by different firmicutes, by mining the genome sequences of all sequenced members of the firmicute phylum. Within this phylum, we also predicted the potential of 18 bacterial strains to be lanthipeptide-producers.Type II toxin-antitoxin (TA) families of Geobacillus strains, which have not been well-studied, have also been covered in this thesis, and 15 putative novel toxins and antitoxins have been identified together with potentially new TA families. Moreover, a hypothesis on the regulation of gene expression of the XRE-COG2856 TA family has been proposed.Overall, the results indicated that Geobacillus sp. ZGt-1 is a source of putative novel antimicrobial peptides and proteins. This study represents the first report on a Geobacillus strain potentially producing a group of various antibacterial peptides and proteins. The results also indicate that members of the thermophilic genus Geobacillus, in general, represent promising producers of antimicrobials.

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