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

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.

Enhancement of Membrane Vesicle Production by Disrupting the degP Gene in Meiothermus Ruber H328

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 DdegP 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 DdegP exhibited smooth growth but a lower level of turbidity at 60ºC although there was no difference in growth at 55ºC between the wild strain and the mutant strain. Finally, we have confirmed that incubation at 60°C increases MV in the mutant strain DdegP 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.

Molecular characterization of a prolyl endopeptidase from a feather-degrading thermophile Meiothermus ruber H328

Prolyl endopeptidase from an aerobic and Gram-negative thermophile Meiothermus ruber H328 (MrPEP) was purified in native and recombinant forms, but both preparations had comparable characteristics. Production of the native MrPEP was increased 10-fold by adding intact chicken feathers. The gene for MrPEP (mrH_2860) was cloned from the genome of strain H328 and found to have no signal sequence at the N-terminus. MrPEP is composed of two major domains: the β-propeller domain and the peptidase domain with a typical active site motif and catalytic triad. Based on extensive investigations with different types of peptide substrates and FRETS-25Xaa libraries, MrPEP showed strict preferences for Pro residue at the P1 position but broader preferences at the P2 and P3 positions in substrate specificity with stronger affinity for residues at the P3 position of substrate peptides that are longer than four residues in length. In conclusion, the molecular characterization of MrPEP resembles its animal counterparts more closely than bacterial counterparts in function and structure.

Molecular and biochemical characterization of a new thermostable bacterial laccase from Meiothermus ruber DSM 1279

A new bacterial laccase gene (mrlac) fromMeiothermus ruberDSM 1279 was successfully overexpressed to produce a laccase (Mrlac) in soluble form inEscherichia coliduring simultaneous overexpression of a chaperone protein (GroEL/ES).