Browsing by Subject "hypothetical proteins of unknown function"

The ever-increasing spread of antibiotic resistant bacteria creates a constant demand for new sources for antimicrobial drugs. Phages are a natural source for antibacterial proteins, but also produce a variety of unknown compounds, referred to as “hypothetical proteins of unknown function” (HPUF). HPUFs usually consist of structural proteins, but also small polypeptides that inhibit bacterial growth during infection. These peptides could be utilized in the discovery of new antimicrobial molecules. However, the current methods used for the screening of such proteins are time consuming and unreliable, making this a fairly unpopular option to utilize. In this study, a new NGS (Next Generation Sequencing) based assay for the screening of phage derived bacteriotoxic proteins was developed and tested by performing two separate experiments together with a previously used plating assay as a comparative method. A preliminary experiment was performed as a proof of principle, with five known toxic and five non-toxic genes. After this, the methods were compared by screening 23 previously identified HPUF genes of phage fHy-Eco03. In the plating assay genes were screened individually by observing growth of bacterial transformants upon gene expression. In the NGS assay genes we screened simultaneously by transforming them to E. coli cells as a pooled sample. Results were obtained with bioinformatics. Toxic genes were expected to be identified through a decrease in sequence read amount, as a consequence of bacterial growth inhibition. In the pre-experiment a difference between toxic and non- toxic proteins was not observed. The results between the NGS and plating assay in the screening of phage fHy-Eco03 genes, were similar and resulted in the identification of one toxic protein. The inconsistent results are probably an outcome of lac promoter repression by glucose supplementation, thus only highly toxic genes show an inhibitory effect. Despite this the NGS assay outperformed the plating assay in both accuracy and efficiency. The NGS assay has high potential to be used as a screening assay for phage derived toxic genes, however further optimization and validation is required, by firstly selecting compatible media and secondly by re- testing with different phages and host bacteria.