Figure 1.

Overview of metasecretome library construction and selection. (A) A shotgun metagenomic library was constructed by cloning metagenomic DNA into the pIII cloning cassette of pDJ01 phagemid vector that does not contain a signal sequence. A small proportion of metagenomic inserts contain signal sequences or other membrane-targeting sequence motifs (red oval shape). (B) Recombinant phagemids replicate as plasmids inside the cells, or alternatively, in the presence of the helper phage, they are packaged as recombinant virions called phagemid particles (PPs). (C) After infection of the library with the gIII-deleted helper phage VCSM13d3, the PPs derived from the recombinant clones that do not contain a membrane-targeting sequence lack the pIII-made cap structure (bottom end of the metagenome phage in the figure). In contrast, the PPs derived from the recombinant phagemids that encode a membrane-targeting sequence in frame with pIII contain the cap structure formed by insert-pIII fusion. Due to the lack of the pIII virion cap, the PPs that do not encode membrane-targeting signals were disassembled in the presence of ionic detergent sarcosyl (Sarcosyl^S), while the secretome protein-displaying PPs were resistant to sarcosyl (Sarcosyl^R), and this was used as a basis for selection. (D) After the removal of ssDNA released from the disassembled Sarcosyl^S PPs, the ssDNA from the intact Sarcosyl^R PPs was purified and used to: (E) transform E. coli to obtain an amplified metasecretome plasmid library for preliminary assessment of metasecretome diversity by Sanger sequencing of clone inserts and (F) as a template for metasecretome analysis by next-generation sequencing.