28 research outputs found
Development and implementation of rapid metabolic engineering tools for chemical and fuel production in Geobacillus thermoglucosidasius NCIMB 11955
Background
The thermophile Geobacillus thermoglucosidasius has considerable attraction as a chassis for the production of chemicals and fuels. It utilises a wide range of sugars and oligosaccharides typical of those derived from lignocellulose and grows at elevated temperatures. The latter improves the rate of feed conversion, reduces fermentation cooling costs and minimises the risks of contamination. Full exploitation of its potential has been hindered by a dearth of effective gene tools.
Results
Here we designed and tested a collection of vectors (pMTL60000 series) in G. thermoglucosidasius NCIMB 11955 equivalent to the widely used clostridial pMTL80000 modular plasmid series. By combining a temperature-sensitive replicon and a heterologous pyrE gene from Geobacillus kaustophilus as a counter-selection marker, a highly effective and rapid gene knock-out/knock-in system was established. Its use required the initial creation of uracil auxotroph through deletion of pyrE using allele-coupled exchange (ACE) and selection for resistance to 5-fluoroorotic acid. The turnaround time for the construction of further mutants in this pyrE minus strain was typically 5 days. Following the creation of the desired mutant, the pyrE allele was restored to wild type, within 3 days, using ACE and selection for uracil prototrophy. Concomitant with this process, cargo DNA (pheB) could be readily integrated at the pyrE locus. The system’s utility was demonstrated through the generation in just 30 days of three independently engineered strains equivalent to a previously constructed ethanol production strain, TM242. This involved the creation of two in-frame deletions (ldh and pfl) and the replacement of a promoter region of a third gene (pdh) with an up-regulated variant. In no case did the production of ethanol match that of TM242. Genome sequencing of the parental strain, TM242, and constructed mutant derivatives suggested that NCIMB 11955 is prone to the emergence of random mutations which can dramatically affect phenotype.
Conclusions
The procedures and principles developed for clostridia, based on the use of pyrE alleles and ACE, may be readily deployed in G. thermoglucosidasius. Marker-less, in-frame deletion mutants can be rapidly generated in 5 days. However, ancillary mutations frequently arise, which can influence phenotype. This observation emphasises the need for improved screening and selection procedures at each step of the engineering processes, based on the generation of multiple, independent strains and whole-genome sequencing
Sixteen diverse laboratory mouse reference genomes define strain-specific haplotypes and novel functional loci.
We report full-length draft de novo genome assemblies for 16 widely used inbred mouse strains and find extensive strain-specific haplotype variation. We identify and characterize 2,567 regions on the current mouse reference genome exhibiting the greatest sequence diversity. These regions are enriched for genes involved in pathogen defence and immunity and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. We used these genomes to improve the mouse reference genome, resulting in the completion of 10 new gene structures. Also, 62 new coding loci were added to the reference genome annotation. These genomes identified a large, previously unannotated, gene (Efcab3-like) encoding 5,874 amino acids. Mutant Efcab3-like mice display anomalies in multiple brain regions, suggesting a possible role for this gene in the regulation of brain development
Identification and structural analysis of the tripartite α-pore forming toxin of Aeromonas hydrophila
The alpha helical CytolysinA family of pore forming toxins (α-PFT) contains single, two, and three component members. Structures of the single component Eschericia coli ClyA and the two component Yersinia enterolytica YaxAB show both undergo conformational changes from soluble to pore forms, and oligomerization to produce the active pore. Here we identify tripartite α-PFTs in pathogenic Gram negative bacteria, including Aeromonas hydrophila (AhlABC). We show that the AhlABC toxin requires all three components for maximal cell lysis. We present structures of pore components which describe a bi-fold hinge mechanism for soluble to pore transition in AhlB and a contrasting tetrameric assembly employed by soluble AhlC to hide their hydrophobic membrane associated residues. We propose a model of pore assembly where the AhlC tetramer dissociates, binds a single membrane leaflet, recruits AhlB promoting soluble to pore transition, prior to AhlA binding to form the active hydrophilic lined pore
The Bacillus cereus Hbl and Nhe Tripartite Enterotoxin Components Assemble Sequentially on the Surface of Target Cells and Are Not Interchangeable
Synthetic DNA and biosecurity: Nuances of predicting pathogenicity and the impetus for novel computational approaches for screening oligonucleotides
NLRP1 restricts butyrate producing commensals to exacerbate inflammatory bowel disease
Anti-microbial signaling pathways are normally triggered by innate immune receptors when detecting pathogenic microbes to provide protective immunity. Here we show that the inflammasome sensor Nlrp1 aggravates DSS-induced experimental mouse colitis by limiting beneficial, butyrate-producing Clostridiales in the gut. The colitis-protective effects of Nlrp1 deficiency are thus reversed by vancomycin treatment, but recapitulated with butyrate supplementation in wild-type mice. Moreover, an activating mutation in Nlrp1a increases IL-18 and IFNÎł production, and decreases colonic butyrate to exacerbate colitis. We also show that, in patients with ulcerative colitis, increased NLRP1 in inflamed regions of the colon is associated with increased IFN-Îł. In this context, NLRP1, IL-18 or IFN-Îł expression negatively correlates with the abundance of Clostridiales in human rectal mucosal biopsies. Our data identify the NLRP1 inflammasome to be a key negative regulator of protective, butyrate-producing commensals, which therefore promotes inflammatory bowel disease