SummaryBacterial ATPases belonging to the ParA family assure partition of their replicons by forming dynamic assemblies which move replicon copies into the new cell-halves. The mechanism underlying partition is not understood for the Walker-box ATPase class, which includes most plasmid and all chromosomal ParAs. The ATPases studied both polymerize and interact with non-specific DNA in an ATP-dependent manner. Previous work showed that in vitro, polymerization of one such ATPase, SopA of plasmid F, is inhibited by DNA, suggesting that interaction of SopA with the host nucleoid could regulate partition. In an attempt to identify amino acids in SopA that are needed for interaction with non-specific DNA, we have found that mutation of codon 340 (lysine to alanine) reduces ATP-dependent DNA binding > 100-fold and correspondingly diminishes SopA activities that depend on it: inhibition of polymer formation and persistence, stimulation of basal-level ATP hydrolysis and localization over the nucleoid. The K340A mutant retained all other SopA properties tested except plasmid stabilization; substitution of the mutant SopA for wild-type nearly abolished mini-F partition. The behaviour of this mutant indicates a causal link between interaction with the cell's non-specific DNA and promotion of the dynamic behaviour that ensures F plasmid partition.

Dormant bacterial spores are encased in a thick protein shell, the “coat”, which contains ~70 different proteins. The coat protects the spore from environmental insults, and is among the most durable static structures in biology. Due to extensive cross-linking among coat proteins, this structure has been recalcitrant to detailed biochemical analysis, so molecular details of how it assembles are largely unknown. Here, we reconstitute the basement layer of the coat atop spherical membranes supported by silica beads to create artificial spore-like particles. We report that these synthetic spore husk-encased lipid bilayers (SSHELs) assemble and polymerize into a static structure, mimicking in vivo basement layer assembly during sporulation in Bacillus subtilis. Additionally, we demonstrate that SSHELs may be easily covalently modified with small molecules and proteins. We propose that SSHELs may be versatile display platforms for drugs and vaccines in clinical settings, or for enzymes that neutralize pollutants for environmental remediation.

The SufBCD complex is an essential component of the SUF machinery of [Fe-S] cluster biogenesis in many organisms. We show here that in Mycobacterium tuberculosis the formation of this complex is dependent on the protein splicing of SufB, suggesting that this process is a potential new target for antituberculous drugs.The worldwide recrudescence of tuberculosis has been associated with the emergence of multidrug-resistant strains of Mycobacterium tuberculosis, its causative agent. This alarming situation has reinforced the need for the urgent development of new antituberculous drugs targeting novel and specific mycobacterial functions.In a recent work (7), we have identified the M. tuberculosis SUF (mobilization of sulfur) machinery as the unique and essential system of [Fe-S] cluster assembly in mycobacteria. This system is required for the maturation of physiologically important metalloproteins and plays an important role in the resistance to iron limitation and oxidative stress. It is encoded by a mycobacterial operon of seven genes, Rv1460 to Rv1466 (according to the M. tuberculosis genome annotation [5]), among which Rv1461 encoding the highly conserved SufB protein (7) is interrupted by an intein coding sequence (15).In the present study, we show the inability of the unspliced SufB protein to play its role in the SUF machinery owing to its inability to interact with some of its Suf partners. This highlights the prerequisite of protein splicing in SufB maturation and validates the SufB protein splicing as a specific molecular target for the development of novel antituberculous drugs since blocking the protein splicing process of essential proteins was proposed as a singular way to efficiently kill mycobacteria (1,3,6,14).Construction of a sufB mutant and expression of unspliced SufB. The Rv1461 open reading frame (ORF), encoding the M. tuberculosis SufB protein, was cloned in pGADT7 and pGBKT7 vectors (Clontech) for yeast two-hybrid assays (7). In these constructs, the Rv1461 gene was mutated in order to block the protein splicing process of the SufB precursor peptide: the asparagine residue at the C-terminal extremity of the intein sequence (position 611) and the adjacent cysteine (i.e., the first residue of the C-extein at position 612) were replaced by an aspartic acid and a valine, respectively. Site-directed mutagenesis was done using complementary oligonucleotide pairs (5Ј-TTGTAGATCGGTGCGGTGACGTCGTGCACGGCGAA CCCGT-3Ј and 5Ј-ACGGGTTCGCCGTGCACGACGTCAC CGCACCGATCTACAA-3Ј). To verify the protein splicing of the recombinant wild-type mycobacterial SufB protein when expressed in yeast and the blockage effect of the mutation, Saccharomyces cerevisiae strain AH109 (Clontech) was electrotransformed with the wild-type and mutated pGADT7 and pGBKT7 derivatives, plated, and grown at 30°C in minimal DOBA (dropout base with agar; BIO101) medium containing amino acid complement (Complete Supplement Mixture; BIO101) devoid of leucine (Leu) or tryptophan (Trp), to select transformed yeast cells. Two milliliters of a 1...

Spores of Bacillus subtilis are dormant cell types that are formed when the bacterium encounters starvation conditions. Spores are encased in a shell, termed the coat, which is composed of approximately seventy different proteins and protects the spore’s genetic material from environmental insults. The structural component of the basement layer of the coat is an exceptional cytoskeletal protein, termed SpoIVA, which binds and hydrolyzes ATP. ATP hydrolysis is utilized to drive a conformational change in SpoIVA that leads to its irreversible self-assembly into a static polymer in vitro. Here, we characterize the middle domain of SpoIVA, the predicted secondary structure of which resembles the chemotaxis protein CheX but, unlike CheX, does not harbor residues required for phosphatase activity. Disruptions in this domain did not abolish ATP hydrolysis, but resulted in mislocalization of the protein and reduction in sporulation efficiency in vivo. In vitro, disruptions in this domain prevented the ATP hydrolysis-driven conformational change in SpoIVA required for polymerization and led to the aggregation of SpoIVA into particles that did not form filaments. We propose a model in which SpoIVA initially assumes a conformation in which it inhibits its own aggregation into particles, and that ATP hydrolysis remodels the protein so that it assumes a polymerization-competent conformation.

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present in the membrane. We have performed contrast-matching small-angle neutron scattering (SANS) experiments to study the conformational changes of the glycosylated form of A1AT for different concentrations of the osmolyte poly(ethelene glycol) (PEG) and in the presence of two different lipid membranes: POPC and POPS. We also monitor the structural changes of the lipid vesicles in the presence of A1AT by SANS. Guinier fits were used as a first approximation to obtain the radius of gyration (Rg) of A1AT. Bragg peaks were used to study structural changes of lipid vesicles. We observed that the Rg of A1AT changes as a function of PEG concentration in solution and when in the presence of lipid vesicles. The deformations monitored through changed in A1AT Rg in the presence of lipid vesicles are compared to the deformations of the glycoprotein observed under osmotic pressure and to the structural changes observed in the lipid vesicles. [1] Petrusca, et al., JBC 2010.