MvaT from Pseudomonas aeruginosa is a member of the histone-like nucleoid structuring protein (H-NS) family of nucleoid-associated proteins widely spread among Gram-negative bacteria that functions to repress the expression of many genes. Recently, it was reported that H-NS from Escherichia coli can form rigid nucleoproteins filaments on DNA, which are important for their gene-silencing function. This raises a question whether the gene-silencing function of MvaT, which has only ∼18% sequence similarity to H-NS, is also based on the formation of nucleoprotein filaments. Here, using magnetic tweezers and atomic force microscopy imaging, we demonstrate that MvaT binds to DNA through cooperative polymerization to form a nucleoprotein filament that can further organize DNA into hairpins or higher-order compact structures. Furthermore, we studied DNA binding by MvaT mutants that fail to repress gene expression in P. aeruginosa because they are specifically defective for higher-order oligomer formation. We found that, although the mutants can organize DNA into compact structures, they fail to form rigid nucleoprotein filaments. Our findings suggest that higher-order oligomerization of MvaT is required for the formation of rigid nucleoprotein filaments that silence at least some target genes in P. aeruginosa . Further, our findings suggest that formation of nucleoprotein filaments provide a general structural basis for the gene-silencing H-NS family members.

The compaction of DNA by multivalent cations has been the subject of many investigations, not only because it is biologically important, but also because it poses a fascinating challenge to our understanding of semiflexible polymers. [1][2][3][4][5] Polyelectrolyte theory has figured out that the attractive potential that leads to DNA compaction originates from correlated fluctuation of counterions shared between DNA segments. 6 Experimental observations have revealed that DNA condensates have generally a toroidal geometry with a typical size of ∼100 nm in diameter. It is generally believed that DNA within a toroid is organized in a hexagonal close-packed lattice, 7 but how such a structure is formed is still not fully understood.Single-molecule measurements have proven helpful to understanding the nucleation and growth of DNA condensates. [8][9][10][11][12][13] A few recent experiments concentrated on the kinetics of DNA compaction. They showed that DNA condenses continuously and linearly with time once the compaction process begins. [14][15][16][17] The results seemed to support the widely accepted opinion that the toroid is formed by continuously absorbing DNA to a primary loop randomly nucleated on DNA. The temporal resolution of the experiments was, however, relatively low. They might not be able to resolve the time trajectories of the DNA compaction.Exerting forces on DNA is a useful way to study processes relevant to DNA. 18 The force may slow down the dynamical process so that details can be observed using an apparatus of finite temporal resolution. Here we report single-molecule studies on the dynamics of hexaammine cobalt chloride-induced DNA compaction under tension. It turns out that the compaction process is more sophisticated than the static structural model has suggested. DNA condenses into toroid in a quantized manner.The measurements were performed using transverse magnetic tweezers similar to the one recently developed by Yan and Marko. 19 Two micrometer-sized beads are tethered to the two ends of a λ-phage DNA (16.4 µm). One bead is fixed in space by a micropipette, and the other is free in solution. A magnetic rod is inserted into the solution to generate a constant force to the free magnetic bead. The experiments were done in phosphate-buffered saline (10 mM PBS, 5 mM NaCl, pH ) 7.4). After adjusting the force on DNA, 10 µL of 10 mM hexaammine cobalt chloride solution was added into the sample cell. The final concentration of the trivalent cations was 100 µM. Figure 1a shows the time course of DNA compaction at a force F ) 0.5 pN. Time courses measured at other concentrations of the trivalent cations (from 35 to 200 µM) are quite similar. After addition of the cations, a long induction period was observed before the compaction started. The induction time becomes longer when a less concentrated solution of trivalent cations is added. The compaction is discontinuous and stepwise. The two big steps in Figure 1a consist of multiple small steps. Such discontinuous compactions were also observed...

Background UDP-glycosyltransferase (UGT) is an important biotransformation superfamily of enzymes. They catalyze the transfer of glycosyl residues from activated nucleotide sugars to acceptor hydrophobic molecules, and function in several physiological processes, including detoxification, olfaction, cuticle formation, pigmentation. The diversity, classification, scaffold location, characteristics, phylogenetics, and evolution of the superfamily of genes at whole genome level, and their association and mutations associated with pyrethroid resistance are still little known. Methods The present study identified UGT genes in Anopheles sinensis genome, classified UGT genes in An. sinensis , Anopheles gambiae , Aedes aegypti and Drosophila melanogaster genomes, and analysed the scaffold location, characteristics, phylogenetics, and evolution of An. sinensis UGT genes using bioinformatics methods. The present study also identified the UGTs associated with pyrethroid resistance using three field pyrethroid-resistant populations with RNA-seq and RT-qPCR, and the mutations associated with pyrethroid resistance with genome re-sequencing in An. sinensis . Results There are 30 putative UGTs in An. sinensis genome, which are classified into 12 families (UGT301, UGT302, UGT306, UGT308, UGT309, UGT310, UGT313, UGT314, UGT315, UGT36, UGT49, UGT50) and further into 23 sub-families. The UGT308 is significantly expanded in gene number compared with other families. A total of 119 UGTs from An. sinensis , An. gambiae , Aedes aegypti and Drosophila melanogaster genomes are classified into 19 families, of which seven are specific for three mosquito species and seven are specific for Drosophila melanogaster . The UGT308 and UGT302 are proposed to main families involved in pyrethroid resistance. The AsUGT308D3 is proposed to be the essential UGT gene for the participation in biotransformation in pyrethroid detoxification process, which is possibly regulated by eight SNPs in its 3′ flanking region. The UGT302A3 is also associated with pyrethroid resistance, and four amino acid mutations in its coding sequences might enhance its catalytic activity and further result in higher insecticide resistance. Conclusions This study provides the diversity, phylogenetics and evolution of UGT genes, and potential UGT members and mutations involved in pyrethroid resistance in An. sinensis , and lays an important basis for the better understanding and further research on UGT function in defense against insecticide stress. ...