Figure Legends
Fig. 1 Overexpression of wspR encoding diguanylate cyclase from P. aeruginosa in ZM4 (ZM4/wspR) and yhjHencoding phosphodiesterase from E. coli in ZM401 (ZM401/yhjH). (A) Phenotypes observed on the engineered strains cultured in the rich medium, (B) Flocculating efficiency () and intracellular accumulation of c-di-GMP () of the bacterial cells, and (C) Qualitative characterization of extracellular cellulose by Congo red staining. ZM4/pHW20 and ZM401/pHW20: ZM4 and ZM401 engineered with the empty vector pHW20a as the control.
Fig. 2 Manipulations of ZMO1055 and impact on self-flocculation of the bacterial cells of Z. mobilis (A), and intracellular accumulation of c-di-GMP (B). + and −: wild-type and mutated genes from ZM4 and ZM401. / and Δ: gene overexpression and deletion. ZM401ΔZMO1055/1055+(AAL) and ZM401ΔZMO1055/1055+(GGAQF): ZM401 manipulated with the deletion of ZMO1055 and overexpression of the recombinant plasmid bearing the doman AAL or GGAQF developed through site-directed mutations in ZMO1055+for the amino acid substitute Ala356Glu and Asp232Ala, respectively. ZM401ΔZMO1055/1055(V526A): ZM401 engineered with the delection of ZMO1055 and overexpression of the recombinant plasmid bearing ZMO1055 with the site-directed reverse mutation for the amino acid substitute Val526Ala. ZM4ΔZMO1055+/1055+(A526V): ZM4 engineered with the delection of ZMO1055+ and overexpression of the recombinant plasmid bearing ZMO1055+ with the site-directed mutation for the amino acid substitute Ala526Val. ZM4/pHW20 and ZM401/pHW20: ZM4 and ZM401 engineered with the empty plasmid pHW20 as the controls.
Fig. 3 Contribution of ZMO0401 to self-flocculation of the bacterial cells of Z. mobilis () and intracellular accumulation of c-di-GMP () (A), and morphologies observed in flask cultures when shaking was stopped for 3−5 seconds (B). / and Δ: gene overexpression and deletion. ZM4/pHW20 and ZM401/pHW20: ZM4 and ZM401 engineered with the empty vector pHW20a as the controls.
Fig. 4 Impact of the deletion (Δ) and overexpression (/) of ZMO1487 (a), ZMO1365 and ZMO0919 (b) on the self-flocculation () of the bacterial cells of Z. mobilis and intracellular accumulation of c-di-GMP (). ZM4/pHW20 and ZM401/pHW20: ZM4 and ZM401 engineered with the empty vector pHW20a as the controls.
Fig. 5 Engineering ZM4 (A) with the deletion (Δ) of ZMO1055, ZMO0401 and ZMO1487, combinationally (), overexpression (/) of ZMO1082-1083, ZMO1082-1084 and ZMO1082-1085 () as well as the deletion of ZMO1055, ZMO0401 and ZMO1487 (1), together with the overexpression of the whole bcs operon ZMO1082-1085 (2), for strategy (1) + (2) to develop the self-flocculating phenotype (), and the morphologies observed in flask culture of the engineered strains (B). ZM4/pHW20: ZM4 engineered with the empty vector pHW20a as the control ().
Fig. 6 Strategies for engineering unicellular Z. mobilis strains with the self-flocculating phenotype through enhancing intracellular accumulation of c-di-GMP to activate cellulose biosynthesis (genes in bold and plain fonts are for deletion and overexpression, respectively).