Enhancement of membrane vesicle production by disrupting the degP gene in Meiothermus ruber H328

Abstract

The phenomenon of membrane vesicle (MV) production is known to be common to all bacterial cells. Although MVs are expected to be employed in a variety of applications, improving MV productivity is essential for applications. Since the deletion of the degP gene, a periplasmic dual-function protease and chaperone, in Escherichia coli has successfully improved MV production capacity, we tried to enhance MV productivity in the thermophilic M. ruber H328 by deleting the degP gene. One gene (mrH_0331) was selected for degP gene from the H328 genome and we constructed the mutant strain ∆degP by deleting the degP gene of the H328 strain that was replaced with the htk gene showing thermophilic kanamaycin resistance by homologous recombination. The mutant strain ∆degP exhibited smooth growth but a lower level of turbidity at 60 °C although there was no difference in growth at 55 °C between the wild strain and the mutant strain. Finally, we have confirmed that incubation at 60 °C increases MV in the mutant strain ∆degP strain about fivefold by using two fluorescent dyes, DiI and FM4-64, which is followed by TEM analysis. The deletion of the degP gene presumably causes an increase in denatured proteins at 60 °C, leading to enhanced MV production. Meanwhile, the S-layer protein included in the outer membrane of the H328 strain increased in the MV fraction prepared from the mutant cells incubated at 60 °C. This indicates that this method is effective for MV production and that degP deletion enhances it in strain H328.