SEARCH

SEARCH BY CITATION

FilenameFormatSizeDescription
mbt212127-sup-0001-si.docx630K

Fig. S1. Verification of the mutants by colony PCR (Lane M: representing standard marker). (A) Single knockout of gene mrcA (kan/1700 bp): Lane 1, JM109 (DE3) as a template; Lane 2, 3 and 4, mrcA as a template; (B) Single knockout of gene mrcB (kan/1700 bp): Lane 1 and 2, JM109 (DE3) as a template; Lane 3 and 4, mrcB as a template; (C) Single knockout of gene pal (cat/1600 bp): Lane 1 and 2, JM109 (DE3) as a template; Lane 3, 4 and 5, pal as a template; (D) Single knockout of gene lpp (kan/1800 bp): Lane 1, 2, 3 and 4, lpp N as a template; Lane 5 JM109 (DE3) as a template; Lane 6, 7 and 8, lpp as a template; (E) Double knockout of genes lpp and mrcB (cat/1200 bp) as well as mrcA and lpp (kan/1800 bp); Lane 1 and 6, others, Lane 3, JM109 (DE3) as a template; Lane 4, lpp mrcB (CmR) as a template (mrcB-vF and mrcB-vR as primers); Lane 5, lpp mrcB (KanR) as a template (mrcB-vF and mrcB-vR as primers); Lane 6, lpp N as a template (lpp-vF and lpp-vR as primers); Lane 7, mrcA lpp (KanR) as a template (lpp-vF and lpp-vR as primers); (F) Double knockout of genes lpp and pal (kan /2100 bp); Lane 1 and 2, lpp pal (KanR) as a template (pal-vF and pal-vR as primers); Lane 3 and 4, (CmR) as a template (pal-vF and pal-vR as primers); Lane 5, nothing; lane 6, JM109 (DE3) as a template; (G) Double knockout of genes pal and mrcB (kan /1700 bp) as well as pal and mrcA (kan/1700 bp); Lane 1, 3 and 5, mrcB pal as a template (pal-vF and pal-vR as primers); Lane 2 and 4, false positive colonies as a template (pal-vF and pal-vR as primers); Lane 6, 7, 8 and 9, JM109 (DE3) as a template; Lane 10, mrcA pal as a template (pal-vF and pal-vR as primers).

Fig. S2. SDS-PAGE analysis of protein samples in TB medium. Lane 1 and 2, the extracellular proteins of the recombinant leaky strains lpp mrcB with pET32a-pth; Lane 3, 4, 5 and 6, the extracellular proteins of the recombinant leaky strains mrcA lpp, lpp, mrcB and mrcA with pET32a-pth; Lane 7, the extracellular proteins of the strains JM109 (DE3) harbouring pET32a-pth; M, protein ladder; Lane 8, 9, 10, 11, 12, 13 and 14, intracellular soluble proteins corresponding to lane 1, 2, 3, 4, 5, 6 and 7.

Fig. S3. SDS-PAGE analysis of protein samples of lpp mrcB with pET22b-rpa in TB medium (M, protein ladder). Lane 1, 2, 3, and 4, the extracellular proteins of the recombinant strains lpp mrcB, mrcB, lpp and JM109 (DE3) harbouring pET22b-rpa; Lane 5, 6, 7 and 8, intracellular soluble proteins corresponding to lane 1, 2, 3 and 4; Lane 9, 10, 11 and 12, intracellular insoluble proteins corresponding to lane 1, 2, 3 and 4.

Fig. S4. Growth curve of the strains in flasks in LB medium. (A) ■, strain lpp mrcB with pET32a-pth; ●, Strain lpp mrcB harbouring pET32a-pth with IPTG induction after four hour cultivation; ▲, Strain mrcA lpp with pET32a-pth. (B) ■, strain mrcB; ●, strain JM109 (DE3).

Table S1. Strains, plasmid and primers used in this study.

Table S2. The sequences of TFs (targeting fragments).

Table S3. The plasmids, primers, resistance markers, targeting fragments and hosts used for mutant construct.

Table S4. Sequences of the PCR products of the mutants of mrcAN, mrcBN and lppN.

Table S5. Growth analysis and protein detection from the recombinant leaky strains with single-gene deletion in TB medium.

Table S6. Growth analysis and protein detection from the recombinant leaky strains with double deletion in TB medium.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.