Assembly of minicellulosomes on the surface of Bacillus subtilis

To cost-efficiently produce biofuels, new methods are needed to convert lignocellulosic biomass into fermentable sugars. One promising approach is to degrade biomass using cellulosomes, which are surface-displayed multicellulase-containing complexes present in cellulolytic Clostridium and Ruminococcus species. In this study we created cellulolytic strains of Bacillus subtilis that display one or more cellulase enzymes. Proteins containing the appropriate cell wall sorting signal are covalently anchored to the peptidoglycan by coexpressing them with the Bacillus anthracis sortase A (SrtA) transpeptidase. This approach was used to covalently attach the Cel8A endoglucanase from Clostridium thermocellum to the cell wall. In addition, a Cel8A-dockerin fusion protein was anchored on the surface of B. subtilis via noncovalent interactions with a cell wall-attached cohesin module. We also demonstrate that it is possible to assemble multienzyme complexes on the cell surface. A three-enzyme-containing minicellulosome was displayed on the cell surface; it consisted of a cell wall-attached scaffoldin protein noncovalently bound to three cellulase-dockerin fusion proteins that were produced in Escherichia coli. B. subtilis has a robust genetic system and is currently used in a wide range of industrial processes. Thus, grafting larger, more elaborate minicellulosomes onto the surface of B. subtilis may yield cellulolytic bacteria with increased potency that can be used to degrade biomass.     

Determining the quality and complexity of next-generation sequencing data without a reference genome

We describe an open-source kPAL package that facilitates an alignment-free assessment of the quality and comparability of sequencing datasets by analyzing k-mer frequencies. We show that kPAL can detect technical artefacts such as high duplication rates, library chimeras, contamination and differences in library preparation protocols. kPAL also successfully captures the complexity and diversity of microbiomes and provides a powerful means to study changes in microbial communities. Together, these features make kPAL an attractive and broadly applicable tool to determine the quality and comparability of sequence libraries even in the absence of a reference sequence. kPAL is freely available at https://github.com/LUMC/kPAL.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0555-3) contains supplementary material, which is available to authorized users.     

Identification of DNA sequence variation in Campylobacter jejuni strains associated with the Guillain-Barré syndrome by high-throughput AFLP analysis

Background  

Campylobacter jejuni is the predominant cause of antecedent infection in post-infectious neuropathies such as the Guillain-Barré (GBS) and Miller Fisher syndromes (MFS). GBS and MFS are probably induced by molecular mimicry between human gangliosides and bacterial lipo-oligosaccharides (LOS). This study describes a new C. jejuni-specific high-throughput AFLP (htAFLP) approach for detection and identification of DNA polymorphism, in general, and of putative GBS/MFS-markers, in particular.     

A new 3D HN(CA)HA experiment for obtaining fingerprint HN-Hα cross peaks in15N- and13C-labeled proteins

Summary A new 3D¹H–¹⁵N–¹³C triple resonance experiment is presented that provides in-phase absorptive cross peaks between amide protons and -protons of the same and the sequentially preceding residue. The experiment yields similar connectivities as those described previously by Montelione and Wagner (1990a) [J. Magn. Reson.,87, 183–188] and Kay et al. (1991) [J. Magn. Reson.,91, 84–92]. However, the pulse sequence was designed to minimize the time that transverse coherence of the¹³C nucleus is present, since this nucleus has the shortest transverse relaxation time of all the nuclei involved in these experiments. This is achieved by using a coherence transfer pathway from¹H^N to¹⁵N,¹³C,¹H and back to the¹H^N. In the sequence described, transverse¹³C coherence is present only for a length of ca. I¹J(C-H). This reduces loss of signal due to transverse relaxation. We tested the technique on uniformly¹⁵N- and¹³C-enriched T4 lysozyme.     

Discovery and structure–activity relationship analysis of Staphylococcus aureus sortase A inhibitors

Methicillin resistant Staphylococcus aureus (MRSA) is a major health problem that has created a pressing need for new antibiotics. Compounds that inhibit the S. aureus SrtA sortase may function as potent anti-infective agents as this enzyme attaches virulence factors to the cell wall. Using high-throughput screening, we have identified several compounds that inhibit the enzymatic activity of the SrtA. A structure–activity relationship (SAR) analysis led to the identification of several pyridazinone and pyrazolethione analogs that inhibit SrtA with IC₅₀ values in the sub-micromolar range. Many of these molecules also inhibit the sortase enzyme from Bacillus anthracis suggesting that they may be generalized sortase inhibitors.