Bis-(3′–5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) is a second messenger known to control a variety of bacterial processes. The model cyanobacterium, Synechocystis sp. PCC 6803, has a score of genes encoding putative enzymes for c-di-GMP synthesis and degradation. However, most of them have not been functionally characterized. Here, we chose four genes in Synechocystis (dgcA–dgcD), which encode proteins with a GGDEF, diguanylate cyclase (DGC) catalytic domain and multiple Per-ARNT-Sim (PAS) conserved regulatory motifs, for detailed analysis. Purified DgcA, DgcB and DgcC were able to catalyze synthesis of c-di-GMP from two GTPs in vitro. DgcA had the highest activity, compared with DgcB and DgcC. DgcD did not show detectable activity. DgcA activity was specific for GTP and stimulated by the divalent cations, magnesium or manganese. Full activity of DgcA required the presence of the multiple PAS domains, probably because of their role in protein dimerization or stability. Synechocystis mutants carrying single deletions of dgcA–dgcD were not affected in their growth rate or biofilm production during salt stress, suggesting that there was functional redundancy in vivo. In contrast, overexpression of dgcA resulted in increased biofilm formation in the absence of salt stress. In this study, we characterize the enzymatic and physiological function of DgcA–DgcD, and propose that the PAS domains in DgcA function in maintaining the enzyme in its active form.
Voltage-dependent K (K) channels control K permeability in response to shifts in the membrane potential. Voltage sensing in K channels is mediated by the positively charged transmembrane domain S4. The best-characterized K channel, KvAP, lacks the distinct hydrophilic region corresponding to the S3-S4 extracellular loop that is found in other K channels. In the present study, we evaluated the topogenic properties of the transmembrane regions within the voltage-sensing domain in KvAP. S3 had low membrane insertion activity, whereas S4 possessed a unique type-I signal anchor (SA-I) function, which enabled it to insert into the membrane by itself. S4 was also found to function as a stop-transfer signal for retention in the membrane. The length and structural nature of the extracellular S3-S4 loop affected the membrane insertion of S3 and S4, suggesting that S3 membrane insertion was dependent on S4. Replacement of charged residues within the transmembrane regions with residues of opposite charge revealed that Asp in S2 and Glu in S3 contributed to membrane insertion of S3 and S4, and increased the stability of S4 in the membrane. These results indicate that the SA-I function of S4, unique among K channels studied to date, promotes the insertion of S3 into the membrane, and that the charged residues essential for voltage sensing contribute to the membrane-insertion of the voltage sensor domain in KvAP.
The physiological effects of caesium (Cs) on living cells are poorly understood. Here, we examined the physiological role of Cs+ on the activity of the potassium transporters in E. coli. In the absence of potassium (K+), Kup-mediated Cs+ uptake partially supported cell growth, however, at a much lower rate than with sufficient K+. In K+-limited medium (0.1 mM), the presence of Cs+ (up to 25 mM) in the medium enhanced growth as much as control medium containing 1 mM K+. This effect depended on the maintenance of basal levels of intracellular K+ by other K+ uptake transporters. Higher amounts of K+ (1 mM) in the medium eliminated the positive effect of Cs+ on growth, and revealed the inhibitory effect of high Cs+ on the growth of wild-type E. coli. Cells lacking Kdp, TrkG and TrkH but expressing Kup grew less well when Cs+ was increased in the medium. A kdp mutant contained an increased ratio of Cs+/K+ in the presence of high Cs+ in the medium and consequently was strongly inhibited in growth. Taken together, under excess Cs+ conditions Kup-mediated Cs+ influx sustains cell growth, which is supported by intracellular K+ supplied by Kdp.
Arabidopsis thaliana contains five tandem-pore domain potassium channels, TPK1-TPK5 and the related one-pore domain potassium channel, KCO3. Although KCO3 is unlikely to be an active channel, it still has a physiological role in plant cells. TPK2 is most similar to KCO3 and both are localized to the tonoplast. However, their function remains poorly understood. Here, taking advantage of the similarities between TPK2 and KCO3, we evaluated Ca 2+ binding to the EF hands in TPK2, and the elements of KCO3 required for K + channel activity. Presence of both EFhand motifs in TPK2 resulted in Ca 2+ binding, but EF1 or EF2 alone failed to interact with Ca 2+ . The EF hands were not required for K + transport activity. EF1 contains two cysteines separated by two amino acids. Replacement of both cysteines with serines in TPK2 increased Ca 2+ binding. We generated a two-pore domain chimeric K + channel by replacing the missing pore region in KCO3 with a pore domain of TPK2. Alternatively, we generated two versions of simple one-pore domain K + channels by removal of an extra region from KCO3. The chimera and one of the simple onepore variants were functional channels. This strongly suggests that KCO3 is not a pseudogene and KCO3 retains components required for the formation of a functional K + channel and oligomerization. Our results contribute to our understanding of the structural properties required for K + channel activity.
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