Uncoupling of nucleotide hydrolysis and polymerization in the ParA protein superfamily disrupts DNA segregation dynamics.

DNA segregation in bacteria is mediated most frequently by proteins of the ParA superfamily that transport DNA molecules attached via the segrosome nucleoprotein complex. Segregation is governed by a cycle of ATP-induced polymerization and subsequent depolymerization of the ParA factor. Here, we establish that hyperactive ATPase variants of the ParA homolog ParF display altered segrosome dynamics that block accurate DNA segregation. An arginine finger-like motif in the ParG centromere-binding factor augments ParF ATPase activity but is ineffective in stimulating nucleotide hydrolysis by the hyperactive proteins. Moreover, whereas polymerization of wild-type ParF is accelerated by ATP and inhibited by ADP, filamentation of the mutated proteins is blocked indiscriminately by nucleotides. The mutations affect a triplet of conserved residues that are situated neither in canonical nucleotide binding and hydrolysis motifs in the ParF tertiary structure nor at interfaces implicated in ParF polymerization. Instead the residues are involved in shaping the contours of the binding pocket so that nucleotide binding locks the mutant proteins into a configuration that is refractory to polymerization. Thus, the architecture of the pocket not only is crucial for optimal ATPase kinetics but also plays a key role in the polymerization dynamics of ParA proteins that drive DNA segregation ubiquitously in procaryotes.

Effect of nitrogen source on cyanophycin synthesis in Synechocystis sp. strain PCC 6308.

Experiments were carried out to examine the effects of nitrogen source on nitrogen incorporation into cyanophycin during nitrogen limitation and repletion, both with or without inhibition of protein synthesis, in cyanobacteria grown on either nitrate or ammonium. The use of nitrate and ammonium, 14N labeled in the growth medium and 15N labeled in the repletion medium, allows the determination of the source of nitrogen in cyanophycin using proton nuclear magnetic resonance spectroscopy. The data suggest that nitrogen from both the breakdown of cellular protein (14N) and directly from the medium (15N) is incorporated into cyanophycin. Nitrogen is incorporated into cyanophycin at different rates and to different extents, depending on the source of nitrogen (ammonium or nitrate) and whether the cells are first starved for nitrogen. These differences appear to be related to the activity of nitrate reductase in cells and to the possible expression of cyanophycin synthetase during nitrogen starvation.