3.1 Role of c-di-GMP in self-flocculation of the bacterial cells
To explore the role of intracellular accumulation of c-di-GMP in the self-flocculation of Z. mobilis , genes wspR andyhjH encoding DGC and PDE in Pseudomonas aeruginosa andEscherichia coli for c-di-GMP biosynthesis and degradation (Güvener et al., 2007; Lindenberg et al., 2013) were overexpressed in ZM4 (ZM4/wspR ) and ZM401 (ZM401/yhjH ), respectively. These manipulations would enhance or compromise the intracellular accumulation of c-di-GMPs in bacterial cells.
While ZM4/wspR self-flocculated incompletely for a loose and flake-like morphology (Fig. 1A) with a flocculating efficiency of 30.9% compared to 6.1% observed in the control ZM4/pHW20a, the self-flocculating phenotype of the control ZM401/pHW20a was disrupted completely, and the flocculation efficiency decreased to 5.5% (Fig. 1B). On the other hand, the intracellular accumulation of c-di-GMP increased drastically to 72.7 pg/mg protein in ZM4/wspR from 10.0 pg/mg proteins detected for the control, but only 1.4 pg (c-di-GMP)/mg protein was detected in ZM401/yhjH , compared to that of 14.3 pg/mg protein detected in the control (Fig. 1B).
Previous studies have confirmed that cellulose fibrils are the chemical basis for the development of the self-flocculating phenotype in ZM401(Xia et al., 2018). Congo red binds to 1,4-β-D-glucopyranosyl units with a strong affinity, which can be used to qualitatively characterize cellulose production qualitatively (Trivedi et al., 2016). When the cultures were inoculated onto agar plates containing the rich medium (RM) supplemented with Congo red and incubated properly, dark red was observed for ZM401/pHW20a due to cellulose production, but a weak staining reaction occurred with ZM4/wspR , ZM4/pHW20a, and ZM401/yhjH , indicating that less cellulose was produced (Fig. 1C). Therefore, intracellular accumulation of c-di-GMP regulates the development of the self-flocculating phenotype in Z. mobilis , but the synthesis of sufficient cellulose by the bacterial cells would be a prerequisite for them to self-flocculate, with significance in industrial applications.
Fig. 1
In the genome of ZM4, ZMO1055, ZMO0401, ZMO1487, ZMO1365 and ZMO0919 are predicted to encode proteins with conserved domains of GGDEF and/or EAL for DGC and/or EAL activities, catalyzing the biosynthesis and degradation of c-di-GMP, respectively (Jones-Burrage et al., 2019). While ZMO1055 and ZMO0401 contain both GGDEF and EAL domains, ZMO1487 contains an EAL domain only, and both ZMO1365 and ZMO0919 contains GGDEF domains (Table 1).
Table 1