Eukarya and, more recently, some bacteria have been shown to rely on a cytoskeleton-based apparatus to drive chromosome segregation. In contrast, the factors and mechanisms underpinning this fundamental process are underexplored in archaea, the third domain of life. Here we establish that the archaeon Sulfolobus solfataricus harbors a hybrid segrosome consisting of two interacting proteins, SegA and SegB, that play a key role in genome segregation in this organism. SegA is an ortholog of bacterial, Walker-type ParA proteins, whereas SegB is an archaea-specific factor lacking sequence identity to either eukaryotic or bacterial proteins, but sharing homology with a cluster of uncharacterized factors conserved in both crenarchaea and euryarchaea, the two major archaeal sub-phyla. We show that SegA is an ATPase that polymerizes in vitro and that SegB is a site-specific DNA-binding protein contacting palindromic sequences located upstream of the segAB cassette. SegB interacts with SegA in the presence of nucleotides and dramatically affects its polymerization dynamics. Our data demonstrate that SegB strongly stimulates SegA polymerization, possibly by promoting SegA nucleation and accelerating polymer growth. Increased expression levels of segAB resulted in severe growth and chromosome segregation defects, including formation of anucleate cells, compact nucleoids confined to one half of the cell compartment and fragmented nucleoids. The overall picture emerging from our findings indicates that the SegAB complex fulfills a crucial function in chromosome segregation and is the prototype of a DNA partition machine widespread across archaea.nucleoid | polymerization | segrosome | Sulfolobus | Walker-type ParA
The mode of action of short, nonhelical antimicrobial peptides is still not well understood. Here we show that these peptides interact with ATP and directly inhibit the actions of certain ATP-dependent enzymes, such as firefly luciferase, DnaK, and DNA polymerase. ␣-Helical and planar or circular antimicrobial peptides did not show such interaction with ATP.
Genome segregation is a fundamental step in the life cycle of every cell. Most bacteria rely on dedicated DNA partition proteins to actively segregate chromosomes and low copy-number plasmids. Here, by employing super resolution microscopy, we establish that the ParF DNA partition protein of the ParA family assembles into a three-dimensional meshwork that uses the nucleoid as a scaffold and periodically shuttles between its poles. Whereas ParF specifies the territory for plasmid trafficking, the ParG partner protein dictates the tempo of ParF assembly cycles and plasmid segregation events by stimulating ParF adenosine triphosphate hydrolysis. Mutants in which this ParG temporal regulation is ablated show partition deficient phenotypes as a result of either altered ParF structure or dynamics and indicate that ParF nucleoid localization and dynamic relocation, although necessary, are not sufficient per se to ensure plasmid segregation. We propose a Venus flytrap model that merges the concepts of ParA polymerization and gradient formation and speculate that a transient, dynamic network of intersecting polymers that branches into the nucleoid interior is a widespread mechanism to distribute sizeable cargos within prokaryotic cells.
The ParA family protein Soj appears to negatively regulate sporulation in Bacillus subtilis by inhibiting transcription from promoters that are activated by phosphorylated Spo0A. We tested in vitro Soj inhibition of Spo0A-independent variants of a promoter that Soj inhibited (PspoIIG). Transcription from the variants was less sensitive to Soj inhibition, suggesting that inhibition of wild-type PspoIIG was linked to transcription activation by Spo0A.Bacillus subtilis responds to nutrient deprivation and high population densities by initiating a pattern of morphological development resulting in the release of a dormant endospore (9,10,12,26). The decision to sporulate is governed by the levels of phosphorylated Spo0A (Spo0AϳP) (9,10,12,26). One central function of Spo0AϳP is to activate the transcription of essential stage II operons, including spoIIA, spoIIE, and spoIIG (2,9,10,24,(26)(27)(28). The spoIIA and spoIIG operons encode sporulation-specific sigma factors that direct early developmental transcription in the forespore and mother cell compartments, respectively (11,15,16,19).The spo0J operon is a dicistronic unit encoding proteins that belong to the ParA and ParB families, whose members are widely dispersed throughout the bacteria (1,7,17). Spo0J is a sequence-specific DNA binding protein in the ParB family that binds in vitro and in vivo to several origin-proximal DNA sites, termed parS (18). Deletion of spo0J results in a phenotype with a low frequency of anucleate cells accumulating during growth and a block to sporulation before stage II (14, 21). The sporulation block is relieved by the deletion of the parA homologue, soj (14). Transcription from spoIIA, spoIIE, and spoIIG promoters is severely reduced in a ⌬spo0J mutant and restored close to wild-type levels in a ⌬soj spo0J double mutant (14,22). Soj associates with the spo0A, spoIIA, spoIIE, and spoIIG promoters in vivo, and these associations are more pronounced in the absence of Spo0J (22,23). In vitro Soj inhibits transcription from the spoIIG promoter (PspoIIG) activated by either Spo0AϳP or the constitutively active C-terminal domain of Spo0A (Spo0AC) (3). These results imply that Soj acts as a negative regulator of sporulation by inhibiting transcription that is activated by phosphorylated Spo0A.In vitro, Soj dissociates complexes formed by incubating a DNA fragment containing PspoIIG, Spo0AC, and RNA polymerase (RNAP) (3). The Soj-induced dissociation of the promoter-Spo0A-RNAP is unusual, and it was suggested that Soj might act at a step in initiation that follows the action of Spo0A (3). To further explore the mechanism of Soj action, we first needed to distinguish whether or not Soj specifically blocked an activity of Spo0A. We reasoned that if Soj targeted a step in transcription that was independent of Spo0A, then Soj inhibition of a PspoIIG variant that had been mutated so it no longer required Spo0A for high activity would be the same as its inhibition of wild-type PspoIIG. To test this idea, we constructed a set of mutated spoIIG pro...
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