Browsing by Author "Juuti, Noora"

Usp45 is the major secreted protein in Lactococcus lactis. Protein’s 27-aa signal peptide (SPUsp45) is widely used for increased secretion and improved yields of heterologous proteins. L. lactis, previously used mainly in food production, has gained increasing popularity in gene technology and, thanks to extensive research, became a Gram-positive model organism alongside Bacillus subtilis. Despite the widespread use of its signal peptide, the biological role of Usp45 protein remains largely a mystery. The aim of this study was to test whether decreased translation of the naturally highly secreted Usp45 protein would lead to improved secretion of desired heterologous proteins. The hypothesis was that high levels of secreted Usp45 cause the Sec translocon becoming a bottleneck and by reducing this strain on the secretion route, the capacity to secrete other proteins could increase. Based on literature, usp45 is not part of the L. lactis core genome and was assumed to be non-vital. To study this, the L. lactis strain NZ9000 was transformed with the plasmid pLEB805 which contains a nisin inducible antisense-usp45 gene (ASusp45) resulting in strain LAC455. The effect of antisense-RNA mediated silencing on growth and morphology of the cells was observed as well as the changes in quantity and quality of secreted proteins in ASusp45 induced cells. The secretion of heterologous proteins was tested with bacteriocins leucocin A and C that were introduced to the cells in expression vectors. This study brought new information on the function of the usp45 gene in L. lactis. The results show that the silencing of the usp45 gene leads to retarded growth rate, multifold ingrowth of the cell wall, aggregation of the cells and the leakage of cytoplasmic proteins leading to loss of viability of the cells. These results demonstrate that Usp45 is vital for the structure of the cell wall, cell separation and normal chain formation, and it probably acts as a vital peptidoglycan hydrolase.