3.2 Endogenous c-di-GMP metabolism in Z. mobilis
Comparative genome analysis between ZM401 and ZM4 detected the SNP mutation in ZMO1055: thymine was replaced by cytosine for the amino acid substitution Ala526Val, and its role in the degradation of c-di-GMP and development of the self-flocculating phenotype in ZM401 has been studied recently (Cao et al., 2022). However, little attention has been paid to the role of wild-type ZMO1055 (ZMO1055+) from ZM4 in the biosynthesis and degradation of c-di-GMP.
When ZMO1055+ was overexpressed in ZM401, its self-flocculating phenotype was disrupted with the flocculating efficiency decreased to 5.0%, and the intracellular accumulation of c-di-GMP decreased drastically to 0.44 pg/mg protein, compared to that of 92.5% and 14.25 pg/mg protein, respectively, observed in the control (Fig. 2). However when ZMO1055 with the SNP mutation from ZM401 (ZMO1055) was overexpressed in ZM401, its self-flocculating phenotype was compromised slightly with the flocculating efficiency decreased to 85.9%, and the intracellular accumulation of c-di-GMP compromised less to 6.07 pg/mg protein, indicating that the SNP mutation substantially mitigated the PDE activity of ZMO1055+ for c-di-GMP degradation, which was supported by the deletion of ZMO1055 from ZM401 for the signal molecule to further increase to 16.68 pg/mg protein (Fig. 2). Manipulation of ZMO1055+ and ZMO1055 in ZM4 through their overexpression and deletion also indicated a strong PDE activity of the wild-type protein for c-di-GMP degradation.
To further confirm the PDE activity, we constructed an expression plasmid carrying ZMO1055+ but with a site-directed mutation of the amino acid substitution Ala356Glu to change the catalytic domain from EAL to AAL, which was experimentally validated to deactivate PDE activity in P. aeruginosa (Kuchma et al., 2007; Nesbitt et al., 2015). When ZMO1055 was knocked out and the recombinant plasmid was transformed into ZM401, no significant change was observed in the intracellular accumulation of c-di-GMP, and the mutant ZM401ΔZMO1055/1055AAL maintained the self-flocculating phenotype (Fig. 2). These experimental results indicate that the substitution of Ala526Val on ZMO1055+ in ZM401 compromised the protein’s PDE activity for c-di-GMP degradation, as exhibited by the native gene bearing the AAL domain. This conclusion was validated by the reverse substitution of Val526Ala in ZM401 (ZM401ΔZMO1055/1055+), as well as the substitution of Ala526Val in ZM4 (ZM4ΔZMO1055+/1055) to compromise the protein’s PDE activity for intracellular accumulation of c-di-GMP to as high as 18.1 pg/mg protein (Fig. 2).
ZMO1055 also contains the GGDEF (GGDQF) domain, which catalyzes the biosynthesis of c-di-GMP. When GGDQF was replaced by GGAQF through the substitution of Asp232Ala to deactivate the protein’s DGC activity in ZM401, the intracellular accumulation of c-di-GMP decreased to 7.82 pg/mg protein, about 50% of that detected in the control, and its self-flocculating phenotype was disrupted completely (Fig. 2). These results indicate the DGC activity of ZMO1055 and its contribution to the biosynthesis of c-di-GMP for the intracellular accumulation of this signal molecule, as well as the development of the self-flocculating phenotype in ZM401.
Fig. 2
ZMO0401 is another protein with the dual domains speculated for the biosynthesis and degradation of c-di-GMP in Z. mobilis . When it was overexpressed, ZM401 lost the self-flocculating phenotype with the flocculating efficiency decreased to 5.6%, and in the meantime extremely low intracellular accumulation of c-di-GMP (0.76 pg/mg protein) was detected, indicating that ZMO0401 might function predominantly on c-di-GMP degradation (Fig. 3). We therefore constructed the knockout mutants ZM401Δ0401 and ZM4Δ0401, respectively, to validate such a speculation.
When ZMO0401 was deleted from ZM401, the intracellular accumulation of c-di-GMP increased to 29.2 from 15.5 pg/mg protein, but no significant change in the self-flocculating phenotype was observed for the mutant, indicating that the intracellular accumulation of c-di-GMP was high enough for activating the biosynthesis of cellulose to flocculate the bacterial cells. As for ZM4, the intracellular accumulation of c-di-GMP increased significantly to 28.2 from 9.8 pg/mg protein when ZMO0401 was deleted, but the flocculating efficiency of ZM4Δ0401 increased slightly to 15.5%, due to its inability to synthesize sufficient cellulose, which we previously showed relies on a mutation in the protein ZMO1082 involved in cellulose production (Cao et al., 2022) (Fig. 3).
Fig. 3
ZMO1487 was predicted to encode a protein with an EAL domain only for c-di-GMP degradation. When ZMO1487 was overexpressed in ZM401 and ZM4, respectively, the intracellular accumulation of c-di-GMP decreased drastically to 0.95 and 0.54 pg/mg protein from 15.48 and 10.04 pg/mg protein detected in the controls, and the self-flocculating phenotype of ZM401 was disrupted (Fig. 4). On the other hand, when ZMO1487 was deleted from ZM4, intracellular accumulation of c-di-GMP increased to 17.62 pg/mg protein, but no significant difference was observed when ZMO1487 was deleted from ZM401 (Fig. 4). The reason for this phenomenon might be due to relatively weak impact of ZMO1487 on c-di-GMP metabolism in ZM401 compared to ZMO1055 and ZMO0401, particularly when the SNP mutation in ZMO1055 substantially compromised its PDE activity and enhanced the intracellular accumulation of c-di-GMP. These experiments indicate the catalytic function of the EAL domain in ZMO1487 on c-di-GMP degradation.
Both ZMO1365 and ZMO0919 were predicted to encode DGC domains for c-di-GMP biosynthesis. Compared to the intracellular accumulation of c-di-GMP at 9.8 pg/mg protein in the control, the overexpression of ZMO1365 and ZMO0919 in ZM4 increased its intracellular accumulation of c-di-GMP to 82.49 and 27.77 pg/mg protein, respectively (Fig. 4B). The extremely high intracellular accumulation of the signal molecule stimulated partial development of the self-flocculating phenotype in the mutants, with their flocculating efficiency increasing to 46.1% and 30.1%, respectively, compared to only 5.5% observed in the control (Fig. 4B). These experimental results validated the catalytic function of the DGC domains in ZMO1365 and ZMO0919 in c-di-GMP biosynthesis.
Fig. 4