A De Novo Robust Clustering Approach for Amplicon-Based Sequence Data.

When analyzing microbial communities, an active and computational challenge concerns the categorization of 16S rRNA gene sequences into operational taxonomic units (OTUs). Established clustering tools use a one pass algorithm to tackle high number of gene sequences and produce OTUs in reasonable time. However, all of the current tools are based on a crisp clustering approach, where a gene sequence is assigned to one cluster. The weak quality of the output compared with more complex clustering algorithms forces the user to postprocess the obtained OTUs. Providing a membership degree when assigning a gene sequence to an OTU will help the user during the postprocessing task. Moreover it is possible to use this membership degree to automatically evaluate the quality of the obtained OTUs. So the goal of this study is to propose a new clustering approach that takes into account uncertainty when producing OTUs, and improves both the quality and the presentation of the OTU results.

Solid-state NMR chemical-shift perturbations indicate domain reorientation of the DnaG primase in the primosome of Helicobacter pylori.

We here investigate the interactions between the DnaB helicase and the C-terminal domain of the corresponding DnaG primase of Helicobacter pylori using solid-state NMR. The difficult crystallization of this 387 kDa complex, where the two proteins interact in a six to three ratio, is circumvented by simple co-sedimentation of the two proteins directly into the MAS-NMR rotor. While the amount of information that can be extracted from such a large protein is still limited, we can assign a number of amino-acid residues experiencing significant chemical-shift perturbations upon helicase-primase complex formation. The location of these residues is used as a guide to model the interaction interface between the two proteins in the complex. Chemical-shift perturbations also reveal changes at the interaction interfaces of the hexameric HpDnaB assembly on HpDnaG binding. A structural model of the complex that explains the experimental findings is obtained.

High Q factor InP photonic crystal nanobeam cavities on silicon wire waveguides.

High-quality (Q) factor indium phosphide (InP)-based 1D photonic crystal nanobeam cavities are fabricated on silicon on insulator waveguides. Through the optimization of the fabrication process, the intrinsic Q factor of these fully encapsulated nanocavities is demonstrated to attain values higher than 100,000. Experimental and numerical investigations are carried out on the impact, on the Q factor, of the strength of the evanescent wave coupling between the cavity and the waveguide. We reveal that this coupling can result in a modification of the electromagnetic field distribution in the resonant mode, which gives rise up to a factor 4 reduction in the intrinsic Q factor for the structures under study.

Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners.

YabA negatively regulates initiation of DNA replication in low-GC Gram-positive bacteria. The protein exerts its control through interactions with the initiator protein DnaA and the sliding clamp DnaN. Here, we combined X-ray crystallography, X-ray scattering (SAXS), modeling and biophysical approaches, with in vivo experimental data to gain insight into YabA function. The crystal structure of the N-terminal domain (NTD) of YabA solved at 2.7 Å resolution reveals an extended α-helix that contributes to an intermolecular four-helix bundle. Homology modeling and biochemical analysis indicates that the C-terminal domain (CTD) of YabA is a small Zn-binding domain. Multi-angle light scattering and SAXS demonstrate that YabA is a tetramer in which the CTDs are independent and connected to the N-terminal four-helix bundle via flexible linkers. While YabA can simultaneously interact with both DnaA and DnaN, we found that an isolated CTD can bind to either DnaA or DnaN, individually. Site-directed mutagenesis and yeast-two hybrid assays identified DnaA and DnaN binding sites on the YabA CTD that partially overlap and point to a mutually exclusive mode of interaction. Our study defines YabA as a novel structural hub and explains how the protein tetramer uses independent CTDs to bind multiple partners to orchestrate replication initiation in the bacterial cell.

Solid-state NMR sequential assignments of the N-terminal domain of HpDnaB helicase.

We present solid-state NMR assignments of the N-terminal domain of the DnaB helicase from Helicobacter pylori (153 residues) in its microcrystalline form. We use a sequential resonance assignment strategy based on three-dimensional NMR experiments. The resonance assignments obtained are compared with automated resonance assignments computed with the ssFLYA algorithm. An analysis of the (13)C secondary chemical shifts determines the position of the secondary structure elements in this α-helical protein.

Subduing surface recombination for continuous-wave operation of photonic crystal nanolasers integrated on Silicon waveguides.

Detrimental surface recombination of carriers in InP-based photonic crystal nanobeams containing quantum wells is reduced by employing chemical treatment followed by silica encapsulation. Carrier lifetime is shown to recover to 2.63ns close to the bulk value. This enables us to obtain optically pumped room-temperature continuous-wave nanolasers at 1.55µm integrated onto Silicon on insulator waveguide platform with a threshold of 8µW.

Structure and primase-mediated activation of a bacterial dodecameric replicative helicase.

Replicative helicases are essential ATPases that unwind DNA to initiate chromosomal replication. While bacterial replicative DnaB helicases are hexameric, Helicobacter pylori DnaB (HpDnaB) was found to form double hexamers, similar to some archaeal and eukaryotic replicative helicases. Here we present a structural and functional analysis of HpDnaB protein during primosome formation. The crystal structure of the HpDnaB at 6.7 Å resolution reveals a dodecameric organization consisting of two hexamers assembled via their N-terminal rings in a stack-twisted mode. Using fluorescence anisotropy we show that HpDnaB dodecamer interacts with single-stranded DNA in the presence of ATP but has a low DNA unwinding activity. Multi-angle light scattering and small angle X-ray scattering demonstrate that interaction with the DnaG primase helicase-binding domain dissociates the helicase dodecamer into single ringed primosomes. Functional assays on the proteins and associated complexes indicate that these single ringed primosomes are the most active form of the helicase for ATP hydrolysis, DNA binding and unwinding. These findings shed light onto an activation mechanism of HpDnaB by the primase that might be relevant in other bacteria and possibly other organisms exploiting dodecameric helicases for DNA replication.

Thermal management in hybrid InP/silicon photonic crystal nanobeam laser.

Thermal properties of InP-based quantum well photonic crystal nanobeam lasers heterogeneously integrated on silicon on insulator waveguides are studied. We show both numerically and experimentally the reduction of the thermal resistance of the III-V cavities by adjusting the composition of the layer which bonds the III-V materials to the silicon wafer and by adding an over-cladding on top of the cavities. Using a bonding layer made of benzocyclobutene and SiO(2) and an over-cladding of MgF(2), we found a decrease by a factor higher than 35 compared to air-suspended photonic crystal nanobeam cavities. Such optimized structures are demonstrated to operate under continuous wave pumping for several 10's of minutes despite the adverse effect of non-radiative surface recombination of carriers.

Experimental demonstration of a hybrid III-V-on-silicon microlaser based on resonant grating cavity mirrors.

We present the experimental demonstration of a novel class of hybrid III-V-on-silicon microlasers. We show that by coupling a silicon cavity to a III-V waveguide, the interaction between the propagating mode in the III-V waveguide and the cavity mode in the silicon resonator results in high, narrowband reflection back into the III-V waveguide, forming a so-called resonant mirror. By combining two such mirrors and providing optical gain in the III-V wire between these two mirrors, laser operation can be realized. This optically pumped device measures 55 by 2 µm, requires microwatt-level threshold pump power, and shows single-mode laser emission with a side-mode suppression ratio of up to 39 dB.

Architecture of a dodecameric bacterial replicative helicase.

Hexameric DnaB helicases are often loaded at DNA replication forks by interacting with the initiator protein DnaA and/or a helicase loader (DnaC in Escherichia coli). These loaders are not universally required, and DnaB from Helicobacter pylori was found to bypass DnaC when expressed in E. coli cells. The crystal structure of Helicobacter pylori DnaB C-terminal domain (HpDnaB-CTD) reveals a large two-helix insertion (named HPI) in the ATPase domain that protrudes away from the RecA fold. Biophysical characterization and electron microscopy (EM) analysis of the full-length protein show that HpDnaB forms head-to-head double hexamers remarkably similar to helicases found in some eukaryotes, archaea, and viruses. The docking of the HpDnaB-CTD structure into EM reconstruction of HpDnaB provides a model that shows how hexamerization of the CTD is facilitated by HPI-HPI interactions. The HpDnaB double-hexamer architecture supports an alternative strategy to load bacterial helicases onto forks in the absence of helicase loaders.