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